Macromolecules

ABSTRACT

A macromolecule includes i) a dendrimer with a core and at least one generation of lysine residue building units, the outermost generation of building units having surface amino groups, ii) a first terminal group covalently attached to a first surface amino group of a building unit, which includes a residue of docetaxel (DTX), and iii) a second terminal group covalently attached to a second surface amino group of a building unit, which includes a pharmacokinetic modifying agent. The pharmacokinetic modifying agent can be a polyethylene glycol (PEG). The first terminal group can be covalently attached to the surface amino group of the dendrimer through a diacid linker. The diacid linker can include a 2,2′-thiodiacetic acid residue. The diacid linker can form an ester bond with a hydroxyl group of the DTX and an amide bond with the surface amino group. A pharmaceutically acceptable salt of the macromolecule can be prepared.

CROSS REFERENCE TO RELATED APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference the sequence listingsubmitted as an ASCII text filed via EFS-Web on Aug. 28, 2017. TheSequence Listing is provided as a file entitled 16796845_1.txt, createdon Dec. 5, 2013, which is 0.6 Kb in size.

FIELD OF THE INVENTION

The present invention relates to a macromolecule comprising a dendrimerhaving surface amine groups to which at least two different, terminalgroups are attached including a pharmaceutically active agent and apharmacokinetic modifying agent, the pharmaceutically active agent beingattached covalently through a diacid linker. Pharmaceutical compositionsand methods of treatment are also described.

BACKGROUND OF THE INVENTION

There are a number of difficulties associated with the formulation anddelivery of pharmaceutically active agents including poor aqueoussolubility, toxicity, low bioavailability, instability under biologicalconditions, lack of targeting to the site of action and rapid in vivodegradation.

To combat some of these difficulties, pharmaceutically active agents maybe formulated with solubilizing agents which themselves may cause sideeffects such as hypersensitivity and may require premedication to reducethese side effects. Alternative approaches include encapsulation of thepharmaceutically active agent in liposomes, micelles or polymer matricesor attachment of the pharmaceutically active agent to liposomes,micelles and polymer matrices.

Although these approaches may improve some of the problems associatedwith the formulation and delivery of pharmaceutically active agents,many still have drawbacks. Oncology drugs can be particularly difficultto formulate and have side effects that may limit the dosage amount andregimen that can be used for treatment. This can result in reducedefficacy of the treatment. For example, taxane drugs such as paclitaxel,docetaxel and cabazitaxel have low aqueous solubility and are oftenformulated with solubilisation 30. excipients such as polyethoxylatedcaster oils (Cremophor EL) or polysorbate 80. Although thesesolubilization excipients allow increased amounts of drug in theformulation, they are known to result in significant side effectsthemselves including hypersensitivity. To reduce hypersensitivity,premedication with steroids such as dexamethasone is sometimes used inthe dosage regimen. However, this also has drawbacks as corticosteroidshave side effects and are not able to be used in diabetic patients,which form a significant subset of patients over 50 with breast cancer.

The use of liposomes, micelles and polymer matrices as carriers eitherencapsulating or having the pharmaceutical agent attached, whileallowing solubilisation of the pharmaceutically active agent and in somecases improved bioavailability and targeting, present difficulties inrelation to release of the pharmaceutically active agent. In some cases,the carrier degrades rapidly releasing the pharmaceutically active agentbefore it has reached the target organ. In other cases, the release ofthe pharmaceutically active agent from the carrier is variable andtherefore may not reach a therapeutic dose of drug in the body or in thetarget organ.

Another difficulty with liposome, micelle and polymer matrices ascarriers is that drug loading can be variable. This can result in somebatches of a particular composition being effective while others are notand/or difficulties in registration of a product for clinical usebecause of variability in the product.

In addition these molecules may be unstable or poorly characterisedmaterials, may suffer from polydispersity, and due to their nature bedifficult to analyse and characterise. They may also have difficultroutes of manufacture. These difficulties, especially with regard toanalysis and batch to batch inconsistency, significantly impede the pathto regulatory submission and approval.

With pharmaceutically active agents that have poor aqueous solubility,often the delivery method is limited, for example, to parenteraladministration. This may limit the dosage regimen available and thedosage that may be delivered.

There is a need for alternative formulations and delivery means fordelivering drugs to reduce side effects, improve dosage regimens andimprove the therapeutic window which may lead to improvements incompliance and efficacy of the drug in patients.

SUMMARY OF THE INVENTION

The invention is predicated in part on the discovery that macromoleculescomprising a dendrimer with surface amino groups having at least twodifferent terminal groups attached to the surface amino groups of thedendrimer and wherein the first terminal group is a pharmaceuticallyactive agent covalently attached to the surface amino group through adiacid linker and the second terminal group is a pharmacokineticmodifying agent may allow high drug loading, improved solubility andcontrolled release of the pharmaceutically active agent.

In a first aspect of the invention there is provided a macromoleculecomprising:

-   -   i) a dendrimer comprising a core and at least one generation of        building units, the outermost generation of building units        having surface amino groups, wherein at least two different        terminal groups are covalently attached to the surface amino        groups of the dendrimer;    -   ii) a first terminal group which is a residue of a        pharmaceutically active agent comprising a hydroxyl group;    -   iii) a second terminal group which is a pharmacokinetic        modifying agent;        wherein the first terminal group is covalently attached to the        surface amino group of the dendrimer through a diacid linker, or        a pharmaceutically acceptable salt thereof.

In some embodiments the pharmaceutically active agent is an oncologydrug, especially docetaxel, paclitaxel, cabazitaxel, camptothecin,topotecan, irinotecan or gemcitabine. In other embodiments thepharmaceutically active agent is a steroid, especially testosterone. Insome embodiments, the pharmaceutically active agent is a sparinglysoluble or insoluble in aqueous solution.

In some embodiments the pharmacokinetic modifying agent is polyethyleneglycol, especially polyethylene glycol having a molecular weight in therange of 220 to 2500 Da, more especially 570 to 2500 Da. In someembodiments, the polyethylene glycol has a molecular weight between 220and 1100 Da, especially 570 and 1100 Da. In other embodiments, thepolyethylene glycol has a molecular weight between 1000 and 5500 Da or1000 and 2500 Da, especially 1000 and 2300 Da.

In some embodiments the diacid linker has the formula:

—C(O)-J-C(O)—X—C(O)—

wherein X is selected from —C₁-C₁₀alkylene-, —(CH₂)_(s)-A-(CH₂)_(t)— andQ; —C(O)-J- is absent, an amino acid residue or a peptide of 2 to 10amino acid residues, wherein the —C(O)— is derived from the carboxyterminal of the amino acid or peptide;A is selected from —O—, —S—, —NR₁—, —N⁺(R₁)₂—, —S—S—, —[OCH₂CH₂]_(r)—O—,—Y—, and —O—Y—O—;Q is selected from Y or —Z═N—NH—S(O)_(w)—Y—;Y is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl;Z is selected from —(CH₂)_(x)—C(CH₃)═, —(CH₂)_(x)CH═, cycloalkyl andheterocycloalkyl;R₁ is selected from hydrogen and C₁-C₄ alkyl;s and t are independently selected from 1 and 2;r is selected from 1, 2 and 3;w is selected from 0, 1 and 2; andx is selected from 1, 2, 3 and 4.

In some embodiments the dendrimer has 1 to 8 generations of buildingunits, especially 3 to 6 generations of building units. In someembodiments the dendrimer is a dendrimer comprising building units oflysine or lysine analogues. In other embodiments the dendrimer comprisesbuilding units of polyetherhydroxylamine.

In some embodiments the first terminal group and the second terminalgroup are present in a 1:1 ratio. In some embodiments the macromoleculecomprises a third terminal group which is a blocking group, especiallyan acyl group such as acetate. In some embodiments the ratio of thefirst terminal group, second terminal group and third terminal group is1:2:1.

In some embodiments, at least 50% of the terminal groups comprise afirst or second terminal group.

In some embodiments the dendrimer comprises a targeting agent attachedto a functional group on the core optionally through a spacer group,especially where the targeting agent is selected from luteinisinghormone releasing hormone, a luteinising hormone releasing hormoneanalog such as deslorelin, LYP-1 and an antibody or fragment thereof.

In some embodiments the macromolecule has a particulate size of lessthan 1000 nm, especially between 5 and 1000 nm, more especially between5 and 400 nm, most especially between 5 and 50 nm. In some embodiments,the macromolecule has a molecular weight of at least 30 kDa, especially40 to 300 kDa, more especially 40 to 150 kDa.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the macromolecule of the invention and apharmaceutically acceptable carrier. In some embodiments, thecomposition is substantially free of solubilisation excipients such aspolyethoxylated caster oils (eg: Cremphor EL) and polysorbate 80. Byremoving the solubilisation excipient the composition of dendrimer isless likely to cause side effects such as acute or delayedhypersensitivity including life-threatening anaphylaxis and/or severefluid retention.

In some embodiments the macromolecule is formulated as a slow-releaseformulation. In some embodiments the linker selected to allowcontrolled-release of pharmaceutically active agent. In someembodiments, the macromolecule is formulated to release greater than 50%of the pharmaceutically active agent in between 5 minutes to 60 minutes.In other embodiments, the macromolecule is formulated to release greaterthan 50% of the pharmaceutically active agent in between 2 hours and 48hours. In yet other embodiments, the macromolecule is formulated torelease greater than 50% of the pharmaceutically active agent in between5 days and 30 days.

In another aspect of the invention there is provided a method oftreating or suppressing the growth of a cancer comprising administeringan effective amount of a macromolecule or pharmaceutical composition ofthe invention in which the pharmaceutically active agent of the firstterminal group is an oncology drug.

In some embodiments, the tumors are primary or metastatic tumors of theprostate, testes, lung, colon, pancreas, kidney, bone, spleen, brain,head and/or neck, breast, gastrointestinal tract, skin or ovary.

In some embodiments, the method comprises administration of acomposition of a macromolecule that is substantially free ofpolyethoxylated caster oils such as Cremophor EL, or polysorbate 80.

In another aspect of the invention there is provided a method ofreducing hypersensitivity upon treatment with an oncology drugcomprising administering a pharmaceutical composition of the presentinvention, wherein the composition is substantially free fromsolubilisation excipients such as Cremophor EL and polysorbate 80.

In a further aspect of the invention there is provided a method ofreducing the toxicity of an oncology drug or formulation of an oncologydrug, comprising administering a macromolecule of the invention in whichthe oncology drug is the pharmaceutically active agent of the firstterminal group.

In some embodiments, the toxicity that is reduced is hematologictoxicity, neurological toxicity, gastrointestinal toxicity,cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity orencephalotoxicity.

In yet a further aspect of the invention there is provided a method ofreducing side effects associated with an oncology drug or formulation ofan oncology drug, comprising administering a macromolecule of theinvention in which the oncology drug is the pharmaceutically activeagent of the first terminal group.

In some embodiments, the side effects which are reduced are selectedfrom neutropenia, leukopenia, thrombocytopenia, myelotoxicity,myelosuppression, neuropathy, fatigue, non-specific neurocognitiveproblems, vertigo, encephalopathy, anemia, dysgeusia, dyspnea,constipation, anorexia, nail disorders, fluid retention, asthenia, pain,nausea, vomiting mucositis, alopecia, skin reactions, myalgia,hypersensitivity and anaphylaxis.

In some embodiments, the need for premedication with agents such ascorticosteroids and anti-histamines is reduced or eliminated.

In yet another aspect of the invention there is provided a method oftreating or preventing a disease or disorder related to low testosteronelevels comprising administering a macromolecule or pharmaceuticalcomposition of the invention in which the pharmaceutically active agentis testosterone.

In some embodiments, the composition is formulated for transdermaldelivery, especially by transdermal patch optionally havingmicroneedles.

DESCRIPTION OF THE INVENTION

A singular forms “a”, “an” and “the” include plural aspects unless thecontext clearly indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein, the term “alkyl” refers to a straight chain or branchedsaturated hydrocarbon group having 1 to 10 carbon atoms. Whereappropriate, the alkyl group may have a specified number of carbonatoms, for example, C₁₋₄alkyl which includes alkyl groups having 1, 2, 3or 4 carbon atoms in a linear or branched arrangement. Examples ofsuitable alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl,3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl,octyl, nonyl and decyl.

The term “alkylene” as used herein refers to a straight-chain divalentalkyl group having 1 to 10 carbon atoms. Where appropriate, the alkylenegroup may have a specified number of carbon atoms, for example C₁-C₆alkylene includes —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅ and—(CH₂)₆—.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated cyclic hydrocarbon. The cycloalkyl ring may include aspecified number of carbon atoms. For example, a 3 to 8 memberedcycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms. Examples ofsuitable cycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentanyl, cyclopentenyl, cyclohexanyl, cyclohexenyl,1,4-cyclohexadienyl, cycloheptanyl and cyclooctanyl.

As used herein, the term “aryl” is intended to mean any stable,monocyclic or bicyclic carbon ring of up to 7 atoms in each ring,wherein at least one ring is aromatic. Examples of such aryl groupsinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl and binaphthyl.

The term “heterocycloalkyl” or “heterocyclyl” as used herein, refers toa cyclic hydrocarbon in which one to four carbon atoms have beenreplaced by heteroatoms independently selected from the group consistingof N, N(R), S, S(O), S(O)₂ and O. A heterocyclic ring may be saturatedor unsaturated. Examples of suitable heterocyclyl groups includetetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl,pyranyl, piperidinyl, pyrazolinyl, dithiolyl, oxathiolyl, dioxanyl,dioxinyl, morpholino and oxazinyl.

The term “heteroaryl” as used herein, represents a stable monocyclic orbityclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and at least one ring contains from 1 to 4 heteroatomsselected from the group consisting of O, N and S. Heteroaryl groupswithin the scope of this definition include, but are not limited to,acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, quinazolinyl,pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, thiophenyl,3,4-propylenedioxythiophenyl, benzothienyl, benzofuranyl, benzodioxane,benzodioxin, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl,imidazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,tetrahydroquinoline, thiazolyl, isothiazolyl, 1,2,4-triazolyl,1,2,3-triazolyl, 1,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,5-triazinyl,1,2,4-triazinyl, 1,2,4,5-tetrazinyl and tetrazolyl.

The term “dendrimer” refers to a molecule containing a core and at leastone dendron attached to the core. Each dendron is made up of at leastone layer or generation of branched building units resulting in abranched structure with increasing number of branches with eachgeneration of building units. The maximum number of dendrons attached tothe core is limited by number of functional groups on the core. The coremay have one or more functional groups suitable to bear a dendron andoptionally an additional functional group for attachment of an agentsuitable for targeting a specific organ or tissue, signalling orimaging.

The term “building unit” used herein refers to a branched moleculehaving at least three functional groups, one for attachment to the coreor a previous generation of building units and at least two functionalgroups for attachment to the next generation of building units orforming the surface of the dendrimer molecule.

The term “generation” refers to the number of layers of building unitsthat make up a dendron or dendrimer. For example, a one generationdendrimer will have one layer of branched building units attached to thecore, for example, Core-[[building unit]]_(u) where u is the number ofdendrons attached to the core. A two generation dendrimer has two layersof building units in each dendron attached to the core, for example,when the building unit has one branch point, the dendrimer may be:Core[[building unit][building unit]₂]_(u), a three generation dendrimerhas three layers of building units in each dendron attached to the core,for example Core-[[building unit][building unit]₂[building unit]₄]_(u),a 6 generation dendrimer has six layers of building units attached tothe core, for example, Core-[[building unit][building unit]₂[buildingunit]₄[building unit]₈[building unit]₁₆[building unit]₃₂]_(u), and thelike. The last generation of building units (the outermost generation)provides the surface functionalisation of the dendrimer and the numberof functional groups available for binding terminal groups. For example,in a dendrimer having a core with two dendrons attached (u=2), if eachbuilding unit has one branch point and there are 6 generations, theoutermost generation has 64 building units and 128 functional groupsavailable to bind terminal groups.

The term “sparingly soluble” as used herein refers to a drug orpharmaceutically active agent that has a solubility between 1 mg/mL and10 mg/mL in water. Drugs that have a solubility in water of less than 1mg/mL are considered insoluble.

The term “pharmaceutically active agent” as used herein refers to acompound that is used to exert a therapeutic effect in vivo. This termis used interchangeably with the term “drug”. The term “residue of apharmaceutically active agent” refers to the portion of themacromolecule that is a pharmaceutically active agent when thepharmaceutically active agent has been modified by attachment to themacromolecule.

The term “oncology drug” used herein refers to a pharmaceutically activeagent used to treat cancer, such as a chemotherapy drug.

As used herein, the term “solubilisation excipient” refers to aformulation additive that is used to solubilize insoluble or sparinglysoluble drugs into an aqueous formulation. Examples include surfactantssuch as polyethoxylated caster oils including Cremophor EL, Cremophor RH40 and Cremophor RH 60, D-α-tocopherol-polyethylene-glycol 1000succinate, polysorbate 20, polysorbate 80, solutol HS 15, sorbitanmonoleate, poloxamer 407, Labrasol and the like.

The macromolecules of the invention may be in the form ofpharmaceutically acceptable salts. It will be appreciated however thatnon-pharmaceutically acceptable salts also fall within the scope of theinvention since these may be useful as intermediates in the preparationof pharmaceutically acceptable salts or may be useful during storage ortransport. Suitable pharmaceutically acceptable salts include, but arenot limited to, salts of pharmaceutically acceptable inorganic acidssuch as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic,benzoic, succinic, oxalic, phenylacetic, methanesulphonic,toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic,glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic,ascorbic and valeric acids.

Base salts include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium.

Basic nitrogen-containing groups may be quarternized with such agents aslower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl and diethylsulfate; and others.

Macromolecules of the Invention

The macromolecules of the invention comprise:

-   -   i) a dendrimer comprising a core and at least one generation of        building units, the outermost generation of building units        having surface amino groups, wherein at least two different        terminal groups are covalently attached to the surface amino        groups of the dendrimer,    -   ii) a first terminal group which is a residue of a        pharmaceutically active agent comprising a hydroxyl group;    -   iii) a second terminal group which is a pharmacokinetic        modifying agent;        wherein the first terminal group is covalently attached to the        surface amino group of the dendrimer through a diacid linker, or        a pharmaceutically acceptable salt thereof.

The dendrimers having surface amino groups have at least two differentterminal groups covalently attached to the surface amino groups.

The first terminal group is a residue of a pharmaceutically active agentcomprising a free hydroxyl group. The pharmaceutically active agent isattached to the surface amino group of the dendrimer through a diacidlinker. The diacid linker forms an ester bond with the hydroxyl group ofthe pharmaceutically active agent and an amide bond with the surfaceamino group.

The pharmaceutically active agent may be any pharmaceutically activeagent that has a hydroxyl group available for ester formation with thediacid linker and is administered to a subject to produce a therapeuticeffect.

In some embodiments the pharmaceutically active agent is an oncologydrug such as a taxane, a nucleoside or a kinase inhibitor, a steroid, anopioid analgesic, a respiratory drug, a central nervous system (CNS)drug, a hypercholesterolemic drug, an antihypertensive drug, animmunosuppressive drug, an antibiotic, a luteinising hormone releasinghormone (LHRH) agonist, a LHRH antagonist, an antiviral drug, anantiretroviral drug, an estrogen receptor modulator, a somatostatinmimic, an anti-inflammatory drug, a vitamin D₂ analogue, a syntheticthyroxine, an antihistamine, an antifungal agent or a nonsteroidalanti-inflammatory drug (NSAID).

Suitable oncology drugs include taxanes such as paclitaxel, cabazitaxeland docetaxel, camptothecin and its analogues such as irinotecan andtopotecan, other antimicrotubule agents such as vinflunine, nucleosidessuch as gemcitabine, cladribine, fludarabine capecitabine, decitabine,azacitidine, clofarabine and nelarabine, kinase inhibitors such assprycel, temisirolimus, dasatinib, AZD6244, AZD1152, PI-103,R-roscovitine, olomoucine and purvalanol A, and epothilone B analoguessuch as Ixabepilone, anthrocyclines such as amrubicin, doxorubicin,epirubicin and valrubicin, super oxide inducers such as trabectecin,proteosome inhibitors such as bortezomib and other topoisomeraseinhibitors, intercalating agents and alkylating agents.

Suitable steroids include anabolic steroids such as testosterone,dihydrotestosterone and ethynylestradiol, and corticosteroids such ascortisone, prednisilone, budesonide, triamcinolone, fluticasone,mometasone, amcinonide, flucinolone, fluocinanide, desonide,halcinonide, prednicarbate, fluocortolone, dexamethasone, betamethasoneand fluprednidine.

Suitable opioid analgesics include morphine, oxymorphone, naloxone,codeine, oxycodone, methylnaltrexone, hydromorphone, buprenorphine andetorphine.

Suitable respiratory drugs include bronchodilators, inhaled steroids,and decongestants and more particularly salbutamol, ipratropium bromide,montelukast and formoterol.

Suitable CNS drugs include antipsychotic such as quetiapine andantidepressants such as venlafaxine.

Suitable drugs to control hypercholesterolemia include ezetimibe andstatins such as simvastatin, lovastatin, atorvastatin, fluvastatin,pitavastatin, provastatin and rosuvastatin.

Suitable antihypertensive drugs include losartan, olmesartan, medoxomil,metrolol, travoprost and bosentan.

Suitable immunosuppressive drugs include glucocorticoids, cytostatics,antibody fragments, anti-immunophilins, interferons, TNF bindingproteins and more particularly, cacineurin inhibitors such astacrolimus, mycophenolic acid and its derivatives such as mycophenolatemofetil, and cyclosporine.

Suitable antibacterial agents include antibiotics such as amoxicillin,meropenem and clavulanic acid.

Suitable LHRH agonists include goserelin acetate, deslorelin andleuprorelin.

Suitable LHRH antagonists include cetrorelix, ganirelix, abarelix anddegarelix.

Suitable antiviral agents include nucleoside analogs such as lamivudine,zidovudine, abacavir and entecavir and suitable antiretroviral drugsinclude protease inhibitors such as atazanavir, lapinavir and ritonavir.

Suitable selective estrogen receptor modulators include raloxifene andfulvestrant.

Suitable somastatin mimics include octreotide.

Suitable anti-inflammatory drugs include mesalazine and suitable NSAIDsinclude acetaminophen (paracetamol).

Suitable vitamin D₂ analogues include paricalcitol.

Suitable synthetic thyroxines include levothyroxine.

Suitable anti-histamines include fexofenadine.

Suitable antifungal agents include azoles such as viriconazole.

In some embodiments the pharmaceutically active agent is sparinglysoluble or insoluble in aqueous solution.

In particular embodiments the pharmaceutically active agent is selectedfrom docetaxel, paclitaxel, testosterone, gemcitabine, camptothecin,irinotecan and topotecan, especially docetaxel, paclitaxel andtestosterone.

The diacid linker that links the pharmaceutically active agent to thesurface amino groups of the dendrimer have the formula:

—C(O)-J-C(O)—X—C(O)—

wherein X is selected from —C₁-C₁₀alkylene-, —(CH₂)_(s)-A-(CH₂)_(t)— andQ;—C(O)-J- is absent, an amino acid residue or a peptide of 2 to 10 aminoacid residues, wherein the —C(O)— is derived from the carboxy terminalof the amino acid or peptide;A is selected from —O—, —S—, —NR₁—, —N⁺(R₁)₂—, —S—S—, —[OCH₂CH₂]_(r)—O—,—Y—, and —O—Y—O—;Q is selected from Y or —Z═N—NH—S(O)_(w)—Y—;Y is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl;Z is selected from —(CH₂)_(x)—C(CH₃)═, —(CH₂)_(x)CH═, cycloalkyl andheterocycloalkyl;R₁ is selected from hydrogen and C₁-C₄ alkyl;s and t are independently selected from 1 and 2;r is selected from 1, 2 and 3;w is selected from 0, 1 and 2; andx is selected from 1, 2, 3 and 4.

In some embodiments one or more of the following applies:

X is —C₁-C₆-alkylene, —CH₂-A-CH₂—, —CH₂CH₂-A-CH₂CH₂— or heteroaryl;—C(O)-J is absent, an amino acid residue or a peptide of 2 to 6 aminoacid residues, wherein the —C(O)— is derived from the carboxy terminalof the amino acid or peptide;A is selected from —O—, —S—, —S—S—, —NH—, —N(CH₃)—, —N⁺(CH₃)₂—,—O-1,2-phenyl-O—, —O-1,3-phenyl-O—, —O-1,4-phenyl-O—, —OCH₂CH₂O—,—[OCH₂CH₂]₂—O— and —[OCH₂CH₂]₃—O—;Y is heteroaryl or aryl, especially thiophenyl,3,4-propylenedioxythiophenyl or benzene;Z is —(CH₂)_(x)C(CH₃)═, —(CH₂)_(x)CH═ and cycloalkyl, especially—CH₂CH₂C(CH₃)═, —CH₂CH₂CH₂C(CH₃)═, —CH₂CH₂CH₂CH═, cyclopentyl andcyclohexyl;R₁ is hydrogen, methyl or ethyl, especially hydrogen or methyl, moreespecially methyl;one of s and t is 1 and the other is 1 or 2, especially were both s andt are 1;r is 1 or 2, especially 2;w is 1 or 2, especially 2; andx is 2 or 1, especially 3.

In some embodiments, —C(O)-J- is absent. In other embodiments, —C(O)-J-is an amino acid residue or a peptide having 2 to 6 amino acid residues.In these embodiments, the N-terminus of the amino acid or peptide formsan amide bond with the —C(O)—X—C(O)— group. In some embodiments, thepeptide is a peptide that comprises an amino acid sequence that isrecognised and cleaved by an endogenous enzyme, such as a protease. Insome embodiments, the enzyme is an intracellular enzyme. In otherembodiments, the enzyme is an extracellular enzyme. In particularembodiments, the enzyme is one that is present in or around neoplastictissue, such as tumor tissue. In some embodiments, the peptide isrecognised by capthesin B or a metalloprotease such as a neutralmetalloproteinase (NMP), MMP-2 and MMP-9. Exemplary peptides includeGGG, GFLG and GILGVP.

In particular embodiments the diacid linker is selected from:

In other embodiments, the diacid linker also comprises a peptide.Exemplary diacid linkers include:

In some embodiments, the diacid linker is selected to provide a desiredrate of release of the drug. For example, a rapid release may berequired where the entire load of pharmaceutical agent is required in ashort space of time whereas a slow release may be more suitable when alow constant therapeutic dose of pharmaceutically active agent isrequired.

In some embodiments, the rate of release is faster than the drugdelivered independent of the macromolecule, especially at least twice asfast. In some embodiments, the drug is released more slowly than thedrug independent of the macromolecule, especially where the drug isreleased at least two times slower, more especially the drug is releasedat least 10 times slower. In some embodiments, the drug is released atleast 30 times slower as described in Example 39. Low rates of releasemay be particularly suitable where the macromolecule includes atargeting group, to enable release of the drug at the active site, butnot in plasma. Low rates of release may also be suitable for drugsformulated to enable slow controlled release delivery over long periodsof time, such as between 1 week and 6 months. The drug may be releasedfrom the macromolecule over a prolonged period of time, such as days,weeks or months. Fast release is preferably release greater than 50%within 0 to 480 minutes, especially within 0 to 120 minutes, and moreespecially within 5 to 60 minutes. Medium release preferably is releasegreater than 50% within 1 to 72 hours, especially within 2 to 48 hours.Slow release is preferably release of greater than 50% in greater than 2days, especially 2 days to 6 months, and more especially within 5 daysto 30 days.

The rate of release of the drug can be controlled by the selection ofthe diacid linker. Diacid linkers containing one or more oxygen atoms intheir backbones, such as diglycolic acid, phenylenedioxydiacetic acid,and polyethylene glycol, or with a cationic nitrogen atom, tend torelease drug at a rapid rate, diacid linkers having one sulfur atom intheir backbone, such as thiodiacetic acid, have a medium rate of releaseand diacid linkers having one or more nitrogen atoms, two or more sulfuratoms, alkyl chains or heterocyclic or heteroaryl groups release thedrug at a slow rate. The rate of release may be summarized by one ormore —O—>—N⁺(R₁)₂—>one —S—>one—NR—>—N—NH—SO₂—>—S—S—>-alkyl->-heterocyclyl-.

It can be seen from Table 2, studies of macromolecules in plasma samplesthat the diglycolic acid (Experiment 3 (b)) released docetaxel at fastrate, with a half life of less than 22 hours, thiodiacetic acid linker(Experiment 8 (c)) released docetaxel at a medium rate, with a half lifeof a little more than 22 hours, extrapolated to around 24 to 30 hoursand the glutaric acid linker (Experiment 5 (b)) released docetaxel at aslow rate with a half life of much greater than 22 hours, and predictedto be more than 2 days. Experiment 16 and 17 do not substantiallyrelease docetaxel in plasma but allow the macromolecule to be targetedto a tumor in which proteases can cleave the peptide sequence to providethe docetaxel at the site of action.

The rate of release may also be dependent on the identity of thepharmaceutically active agent.

In some embodiments, each pharmaceutically active agent is attached tothe dendrimer with the same diacid linker. In other embodiments, two ormore different diacid linkers are used allowing the pharmaceuticallyactive agent to be released from the macromolecule at different rates.

The second terminal group is a pharmacokinetic modifying agent, whichmay be any molecule or residue thereof that modifies or modulates thepharmacokinetic profile of the pharmaceutically active agent or themacromolecule including absorption, distribution, metabolism and/orexcretion. In a particular embodiment, the pharmacokinetic modifyingagent is an agent selected to prolong the plasma half-life of thepharmaceutically active agent, such that the macromolecule has a halflife that is greater than the half-life of the native pharmaceuticallyactive agent, or the marketed pharmaceutically active agent in anon-dendrimer formulation. Preferably the half life of the macromoleculeor composition is at least 2 times and more preferably 10 times greaterthan the native pharmaceutically active agent, or the marketedpharmaceutically active agent in a non-dendrimer formulation.

In some embodiments, the second terminal group is polyethylene glycol(PEG), a polyalkyloxazoline such as polyethyloxazoline (PEOX),polyvinylpyrolidone and polypropylene glycol, especially PEG. In otherembodiments, the second terminal group is a polyether dendrimer.

In some embodiments, the PEG has a molecular weight of between 220 and5500 Da. In some embodiments, the PEG has a molecular weight of 220 to1100 Da, especially 570 and 1100 Da. In other embodiments, the PEG has amolecular weight of 1000 to 5500 Da, especially 1000 to 2500 Da or 1000to 2300.

In some embodiments, the macromolecule comprises a third terminal group.The third terminal group is a blocking group that serves to block thereactivity of a surface amino group of the dendrimer. In particularembodiments, the blocking group is an acyl group such as a C₂-C₁₀ acylgroup, especially acetyl. In other embodiments, the third terminal groupis a second pharmaceutically active agent or a targeting agent.

In some embodiments where there is a first terminal group and a secondterminal group, the ratio of first terminal group and second terminalgroup is between 1:2 and 2:1, especially 1:1.

In some embodiments where there is a first terminal group, a secondterminal group and a third terminal group, the ratio is 1:1:1 to 1:2:2,especially 1:2:1.

In some embodiments, not all of the surface amino groups of thedendrimer are bound to a first terminal group, a second terminal group,or a third terminal group. In some embodiments, some of the surfaceamino groups remain free amino groups. In some embodiments at least 50%of the total terminal groups comprise one of a pharmacokinetic modifyingagent or a pharmaceutically active agent, especially at least 75% or atleast 80% of the terminal groups comprise one of a pharmacokineticmodifying agent or a pharmaceutically active agent. In particularembodiments, a pharmaceutically active agent is bound to greater than14%, 25%, 27%, 30% 39%, 44% or 48% of the total number of surface aminogroups. Where dendrimer is a G5 polylysine dendrimer, the total numberof the pharmaceutically active agent is preferably greater than 15, andespecially greater than 23 and more especially greater than 27. In someembodiments, the pharmacokinetic modifying agent is bound to greaterthan 15%, 25%, 30%, 33% or 46% of the total number of surface aminogroups. Where dendrimer is a G5 polylysine dendrimer, the total numberof pharmacokinetic modifying agents is preferably greater than 25, andespecially greater than 30.

The macromolecule of the invention comprises a dendrimer in which theoutermost generation of building units has surface amino groups. Theidentity of the dendrimer of the macromolecule is not particularlyimportant, provided it has surface amino groups. For example, thedendrimer may be a polylysine, polylysine analogue, polyamidoamine(PAMAM), polyethyleneimine (PEI) dendrimer or polyether hydroxylamine(PEHAM) dendrimer.

The dendrimer comprises a core and one or more dendrons made of one ormore building units. The building units are built up in layers referredto as generations.

In some embodiments, the building unit is a polyamine, more preferably adi or tri-amino with a single carboxylic acid. Preferably the molecularweight of the building unit is from 110 Da to 1 KDa. In someembodiments, the building unit is lysine or lysine analogue selectedfrom:

Lysine 1: having the structure:

Glycyl-Lysine 2 having the structure:

Analogue 3, having the structure below, where a is an integer of 1 or 2;b and c are the same or different and are integers of 1 to 4:

Analogue 4, having the structure below, where a is an integer of 0 to 2;b and c are the same or different and are integers of 2 to 6:

Analogue 5, having the structure below, where a is an integer of 0 to 5;b and c are the same or different and are integers of 1 to 5:

Analogue 6, having the structure below, where a is an integer of 0 to 5;b and c are the same or different and are integers of 0 to 5:

Analogue 7, having the structure below, where a is an integer of 0 to 5;b and c are the same or different and are integers of 1 to 5:

Analogue 8, having the structure below, where a is an integer of 0 to 5;b, c and d are the same or different and are integers of 1 to 5:

Analogue 9, having the structure below, where a is an integer of 0 to 5;b and c are the same or different and are integers of 1 to 5:

and furthermore, the alkyl chain moieties (eg: —C—C—C—) of the buildingunits may be understood to include alkoxy fragments such as C—O—C orC—C—O—C—C where one or more non-adjacent carbon atom is replaced with anoxygen atom, provided that such a substitution does not form a O—C—Xgroup where X is O or N.

In some embodiments the building unit is an amidoamine building unitwith the structure 10:

an etherhydroxyamine building unit with the structure 11:

or a propyleneimine building unit with the structure 12:

In a preferred aspect of the invention, the building units are selectedfrom Lysine 1, Glycyl-Lysine 2 or Lysine analogue 5:

where a is an integer of 0 to 2 or the alkyl link is C—O—C; b and c arethe same or different and are integers of 1 to 2; especially where thebuilding units are lysine.

In some embodiments, the core is a monoamine compound, diamine compound,triamine compound, tetraamine compound or pentamine compound, one ormore of the amine groups having a dendron comprising building unitsattached thereto. In particular embodiments, the molecular weight of thebuilding unit is from 110 Da to 1 KDa.

Suitable cores include benzhydrylamine (BHA), a benzhydrylamide oflysine (BHALys) or a lysine analogue, or:

where a is an integer of 1 to 9, preferably 1 to 5;

where a, b and c, which may be the same or different, and are integersof 1-5, and d is an integer from 0-100, preferably 1-30;

where a and b, may be the same or different, and are integers of 0 to 5;

where a and c, which may be the same or different, are integers of 1 to6 and where c is an integer from 0 to 6;

where a and d, which may be the same or different, are integers of 1 to6 and where b and c, which may be the same or different, are integersfrom 0 to 6;

where a and b are the same or different and are integers of 1 to 5,especially 1 to 3, more especially 1;a triamine compound selected from:

where a, b and c, which may be the same or different, are integers of 1to 6;

where a, b and c, which may be the same or different, are integers of 0to 6;

where a, b and c, which may be the same or different, are integers of 0to 6;

where a, b and c, which may be the same or different, are integers of 0to 6; and d, e and f, which may be the same or different, are integersof 1 to 6;

where a, b and c, which may be the same or different, are integers of 1to 6;

wherein a, b and c, which may be the same or different, are integers of1 to 5, d is an integer from 1 to 100, preferably 1 to 30, e is aninteger from 0 to 5 and f and g are the same or different and areintegers from 1 to 5;or a tetraamine compound selected from

where a, b, c and d, which may be the same or different, are integers of0 to 6;

where a, b, c and d, which may be the same or different, are integers of1 to 6;

where a, b, c and d, which may be the same or different, are integers of0 to 6; and e, f, g and h, which may be the same or different, areintegers of 1 to 6;and furthermore, the alkyl chain moieties (eg: —C—C—C—) of the buildingunits may be understood to include alkoxy fragments such as C—O—C orC—C—O—C—C where one or more non-adjacent carbon atom is replaced with anoxygen atom, provided that such a substitution does not form a O—C—Xgroup where X is O or N.

In some embodiments, the core has at least two amino functional groups,one of which has attached a targeting moiety either directly or througha spacer group. At least one of the remaining functional groups of thecore having a dendron attached as described in WO 2008/017125.

The targeting agent is an agent that binds to a biological target cell,organ or tissue with some selectivity thereby assisting in directing themacromolecule to a particular target in the body and allowing itsaccumulation at that target cell, organ or tissue. The targeting groupmay in addition provide a mechanism for the macromolecule to be activelytaken into the cell or tissue by receptor mediated endocytosis.

Particular examples include lectins and antibodies and other ligands(including small molecules) for cell surface receptors. The interactionmay occur through any type of bonding or association including covalent,ionic and hydrogen bonding, Van der Waals forces.

Suitable targeting groups include those that bind to cell surfacereceptors, for example, the folate receptor, adrenergic receptor, growthhormone receptor, luteinizing hormone receptor, estrogen receptor,epidermal growth factor receptor, fibroblast growth factor receptor (egFGFR2), IL-2 receptor, CFTR and vascular epithelial growth factor (VEGF)receptor.

In some embodiments, the targeting agent is luteinising hormonereleasing hormone (LHRH) or a derivative thereof that binds toluteinising hormone releasing hormone receptor. LHRH has the sequence:pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. Suitable derivatives ofLHRH include those in which one of residues 4-7 are replaced by anotheramino acid, especially residue 6 (Gly). In some embodiments, thereplacement amino acid residue is suitably one that has a side chaincapable of forming a bond with the core or with the spacer. In someembodiments the derivative is LHRH Gly6Lys, LHRH Gly6Asp or LHRHGly6Glu, especially LHRH Gly6Lys. In other embodiments, the derivativeis LHRH Gly6Trp (deslorelin). This receptor is often found oroverexpressed in cancer cells, especially in breast, prostate, ovarianor endometrial cancers.

In some embodiments, the targeting agent is LYP-1, a peptide thattargets the lymphatic system of tumors but not the lymphatic system ofnormal tissue. LYP-1 is a peptide having the sequenceH-Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys-OH and in which the peptide is incyclic form due to a disulfide bond between the sulphur atoms of the twocysteine residues.

In some embodiments, the targeting agent may be an RGD peptide. RGDpeptides are peptides containing the sequence -Arg-Gly-Asp-. Thissequence is the primary integrin recognition site in extracellularmatrix proteins.

Antibodies and antibody fragments such as scFvs and diabodies known tointeract with receptors or cellular factors include CD20, CD52, MUC1,Tenascin, CD44, TNF-R, especially CD30, HER2, VEGF, EGF, EFGR and TNF-α.

In some embodiments the targeting agent may be folate. Folate is avitamin that is essential for the biosynthesis of nucleotide bases andis therefore required in high amounts in proliferating cells. In cancercells, this increased requirement for folic acid is frequently reflectedin an overexpression of the folate receptor which is responsible for thetransport of folate across the cell membrane. In contrast, the uptake offolate into normal cells is facilitated by the reduced folate carrier,rather than the folate receptor. The folate receptor is upregulated inmany human cancers, including malignancies of the ovary, brain, kidney,breast, myeloid cells and the lung and the density of folate receptorson the cell surface appears to increase as the cancer develops.

Estrogens may also be used to target cells expressing estrogen receptor.

The targeting agent may be bound to the dendrimer core directly orpreferably through a spacer. The spacer group may be any divalent groupcapable of binding to both the functional group of the core and thefunctional group on the targeting agent. The size of the spacer group ispreferably sufficient to prevent any steric crowding. Examples ofsuitable spacer groups include alkylene chains and alkylene chains inwhich one or more carbon atoms is replaced by a heteroatom selected from—O—, —S—, or NH. The alkylene chain terminates with functional groupssuitable for attachment to both the core functional group and thetargeting agent. Exemplary spacer groups include X—(CH₂)_(p)—Y,X—(CH₂O)_(p)—CH₂—Y, X—(CH₂CH₂O)_(p)—CH₂CH₂—Y andX—(CH₂CH₂CH₂O)_(p)CH₂CH₂CH₂—Y, where X and Y are functional groups forbinding with or bound to the core and the targeting agent respectively,and p is an integer from 1 to 100, especially 1 to 50 or 1 to 25.

In some embodiments, the targeting group may be bound to the surfaceamino groups as third functional group. In some embodiments, 1 to 32targeting groups are bound to the surface, especially, 1 to 10 arebound, more especially 1 to 4 are bound.

In some embodiments, the targeting agent and the spacer group aremodified to facilitate reaction. For example, the spacer group mayinclude an azide functional group and the targeting agent may include analkyne group or the spacer group is modified with an alkyne and thetargeting agent modified with an azide and the two groups are conjugatedusing a click reaction.

In some embodiments the functional group of the core that does not beara dendron may be bound to biotin, optionally through a spacer groupdescribed above, and the macromolecule reacted with an avidin-antibodyor avidin-biotin-antibody complex. Each avidin complex may bind up to 4macromolecule-biotin conjugates or a combination of macromolecule-biotinconjugates and antibody-biotin conjugates.

In particular embodiments, the core is BHA or BHALys orNEOEOEN[SuN(PN)₂].

In some embodiments, the dendrimer has 1 to 5 dendrons attached to thecore, especially 2 to 4 dendrons, more especially 2 or 3 dendrons.

In some embodiments, the dendrimer has 1 to 8 generations of buildingunits, especially 2 to 7 generations, 3 to 6 generations, moreespecially 4 to 6 generations.

The macromolecule of the invention may be nanoparticulate having aparticulate diameter of below 1000 nm, for example, between 5 and 1000nm, especially 5 and 500 nm, more especially 5 to 400 nm, such as 5 to50 nm, especially between 5 and 20 nm. In particular embodiments, thecomposition contains macromolecules with a mean size of between 5 and 20nm. In some embodiments, the macromolecule has a molecular weight of atleast 30 kDa, for example, 40 to 150 kDa or 40 to 300 kDa.

In some embodiments, the macromolecules of the invention have a particlesize that is suitable for taking advantage of the Enhanced Permeabilityand Retention Effect (EPR effect) in tumors and inflammatory tissue.Blood vessels formed in tumors are formed quickly and are abnormalbecause of poorly-aligned defective endothelial cells, a lack of smoothmuscle layer and/or innervation with a wider lumen. This makes the tumorvessels permeable to particles of a size that would not normally exitthe vasculature and allow the macromolecules to collect in tumor tissue.Furthermore, tumor tissues lack effective lymphatic drainage thereforeonce the macromolecules have entered the tumor tissue, they are retainedthere. Similar accumulation and retention is found in sites ofinflammation.

The macromolecule of the invention may have a loading ofpharmaceutically active agent of 2, 4, 8, 16, 32, 64 or 120 residues,especially 16, 32 or 64 residues per macromolecule.

Methods of making dendrimers are known in the art. For example, thedendrimers of the macromolecule may be made by a divergent method or aconvergent method or a mixture thereof.

In the divergent method each generation of building units issequentially added to the core or an earlier generation. The surfacegeneration having one or both of the surface amino groups protected. Ifone of the amino groups is protected, the free amino group is reactedwith one of the linker, the linker-pharmaceutically active agent or thepharmacokinetic modifying agent. If both amino groups are protected,they are protected with different protecting groups, one of which may beremoved without removal of the other. One of the amino protecting groupsis removed and reacted with one of the linker, thelinker-pharmaceutically active agent or the pharmacokinetic modifyingagent. Once the initial terminal group has been attached to thedendrimer, the other amino protecting group is removed and the other ofthe first and second terminal group is added. These groups are attachedto the surface amino groups by amide formation as known in the art.

In the convergent method, each generation of building units is built upon the previous generation to form a dendron. The first and secondterminal groups may be attached to the surface amino groups as describedabove before or after attachment of the dendron to the core.

In a mixed approach, each generation of building units is added to thecore or a previous generation of building units. However, before thelast generation is added to the dendrimer, the surface amino groups arefunctionalised with terminal groups, for example, a first and secondterminal group, a first and third terminal group or a second and thirdterminal group. The functionalised final generation is then added to thesubsurface layer of building units and the dendron is attached to thecore.

The pharmaceutically active agent is reacted with one of the carboxylicacids of the linker by ester formation as known in the art. For example,an activated carboxylic acid is formed, such as an acid chloride or ananhydride is used and reacted with the hydroxy group of thepharmaceutically active agent. If the pharmaceutically active agent hasmore than one hydroxy group, further hydroxy groups may be protected.

In the case where a targeting agent is attached to the core, afunctional group on the core may be protected during formation of thedendrimer then deprotected and reacted with the targeting agent, thespacer group or the targeting agent-spacer group. Alternatively, thecore may be reacted with the spacer group or targeting agent-spacergroup before the formation of the dendrimer.

Suitable protecting groups, methods for their introduction and removalare described in Greene & Wuts, Protecting Groups in Organic Synthesis,Third Edition, 1999.

Compositions Comprising the Macromolecule

While it is possible that the macromolecules of the invention may beadministered as a neat chemical, in particular embodiments, themacromolecule is presented as a pharmaceutical composition.

The invention provides pharmaceutical formulations or compositions, bothfor veterinary and for human medical use, which comprise one or moremacromolecules of the invention or a pharmaceutically acceptable saltthereof, with one or more pharmaceutically acceptable carriers, andoptionally any other therapeutic ingredients, stabilisers, or the like.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The compositions of the inventionmay also include polymeric excipients/additives or carriers, e.g.,polyvinylpyrrolidones, derivatised celluloses such ashydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin. The compositions may further includediluents, buffers, binders, disintegrants, thickeners, lubricants,preservatives (including antioxidants), flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitanesters, lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, fatty acids and fattyesters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA,zinc and other such suitable cations). Other pharmaceutical excipientsand/or additives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19.sup.th ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), andin “Handbook of Pharmaceutical Excipients”, Third Ed., Ed. A. H. Kibbe,Pharmaceutical Press, 2000.

The macromolecule may also be formulated in the presence of anappropriate albumin protein such as human serum albumin. Albumin carriesnutrients around the body and may bind to the macromolecule and carry itto its site of action.

The macromolecules of the invention may be formulated in compositionsincluding those suitable for oral, rectal, topical, nasal, inhalation tothe lung, by aerosol, ophthalmic, or parenteral (includingintraperitoneal, intravenous, subcutaneous, or intramuscular injection)administration. The compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing themacromolecule into association with a carrier that constitutes one ormore accessory ingredients. In general, the compositions are prepared bybringing the macromolecule into association with a liquid carrier toform a solution or a suspension, or alternatively, bring themacromolecule into association with formulation components suitable forforming a solid, optionally a particulate product, and then, ifwarranted, shaping the product into a desired delivery form. Solidformulations of the invention, when particulate, will typically compriseparticles with sizes ranging from about 1 nanometer to about 500microns. In general, for solid formulations intended for intravenousadministration, particles will typically range from about 1 nm to about10 microns in diameter. The composition may contain macromolecule of theinvention that are nanoparticulate having a particulate diameter ofbelow 1000 nm, for example, between 5 and 1000 nm, especially 5 and 500nm, more especially 5 to 400 nm, such as 5 to 50 nm and especiallybetween 5 and 20 nm. In particular embodiments, the composition containsmacromolecules with a mean size of between 5 and 20 nm. In someembodiments, the macromolecule is polydispersed in the composition, withPDI of between 1.01 and 1.8, especially between 1.01 and 1.5, and moreespecially between 1.01 and 1.2. In particular embodiments, themacromolecule is monodispersed in the composition. Particularlypreferred are sterile, lyophilized compositions that are reconstitutedin an aqueous vehicle prior to injection.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the macromolecule, which canbe formulated to be isotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired macromolecule or a salt thereof.The desired formulation may be placed in a small chamber and nebulized.Nebulization may be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the macromolecules or salts thereof.

Often drugs are co-administered with other drugs in combination therapy,especially during chemotherapy. The macromolecules of the invention maytherefore be administered as combination therapies. For example, whenthe pharmaceutically active agent is docetaxel, the macromolecule may beadministered with doxorubicin, cyclophosphamide or capecitabine. Notonly can the macromolecules be administered with other chemotherapydrugs but may also be administered in combination with other medicationssuch as corticosteroids, anti-histamines, analgesics and drugs that aidin recovery or protect from hematotoxicity, for example, cytokines.

In some embodiments, particularly with oncology drugs, the compositionis formulated for parenteral infusion as part of a chemotherapy regimen.In these embodiments, the compositions are substantially free orentirely free of solubilisation excipients, especially solubilisationexcipients such as Cremophor and polysorbate 80. In particularembodiments, the pharmaceutically active agent is selected fromdocetaxel or paclitaxel and the formulation is substantially free orentirely free of solubilisation excipients such as Cremophor andpolysorbate 80. By removing the solubilisation excipient the compositionof dendrimer is less likely to cause side effects such as acute ordelayed hypersensitivity including life-threatening anaphylaxis and/orsevere fluid retention.

In some embodiments, the macromolecule is formulated for transdermaldelivery such as an ointment, a lotion or in a transdermal patch or useof microneedle technology. High drug loading and aqueous solubilityallows small volumes to carry sufficient drug for patch and microneedletechnologies to provide a therapeutically effective amount. Suchformulations are particularly suitable for delivery of testosterone.

The macromolecules of the invention may also be used to providecontrolled-release of the pharmaceutically active agents and/orslow-release formulations.

In slow-release formulations, the formulation ingredients are selectedto release the macromolecule from the formulation over a prolongedperiod of time, such as days, weeks or months. This type of formulationincludes transdermal patches or in implantable devices that may bedeposited subcutaneously or by injection intravenously, subcutaneously,intramuscularly, intraepidurally or intracranially.

In controlled-release formulations, the diacid linker is selected torelease a majority of its pharmaceutically active agent in a given timewindow. For example, when the time taken for a majority of themacromolecule to accumulate in a target organ, tissue or tumor is known,the linker may be selected to release a majority of its pharmaceuticallyactive agent after the time to accumulate has elapsed. This can allow ahigh drug load to be delivered at a given time point at the site whereits action is required. Alternatively, the linker is selected to releasethe pharmaceutically active agent at a therapeutic level over aprolonged period of time.

In some embodiments, the formulation may have multiplecontrolled-release characteristics. For example, the formulationcomprises macromolecules in which the drug is attached through differentlinkers allowing an initial burst of fast-released drug followed byslower release at low but constant therapeutic levels over a prolongedperiod of time.

In some embodiments, the formulation may have both slow-release andcontrolled-release characteristics. For example, the formulationingredients may be selected to release the macromolecule over aprolonged period of time and the linker is selected to deliver aconstant low therapeutic level of pharmaceutically active agent.

In some embodiments, the pharmaceutically active agent is attached tothe same molecule through different linkers. In other embodiments, eachdrug-linker combination is attached to different macromolecules in thesame formulation.

Methods of Use

The macromolecule of the invention may be used to treat or prevent anydisease, disorder or symptom that the unmodified pharmaceutically activeagent can be used to treat or prevent.

In some embodiments, where the pharmaceutically active agent is anoncology drug, the macromolecule is used in a method of treating orpreventing cancer, or suppressing the growth of a tumor. In particularembodiments, the drug is selected from docetaxel, camptothecin,topotecan, irinotecan and gemcitabine, especially docetaxel.

In some embodiments, the cancer is a blood borne cancer such asleukaemia or lymphoma. In other embodiments, the cancer is a solidtumor. The solid tumor may be a primary or a metastatic tumor. Exemplarysolid tumors include tumors of the breast, lung especially non-smallcell lung cancer, colon, stomach, kidney, brain, head and neckespecially squamous cell carcinoma of the head and neck, thyroid, ovary,testes, liver, melanoma, prostate especially androgen-independent(hormone refractory) prostate cancer, neuroblastoma and gastricadenocarcinoma including adenocarcinoma of the gastroesophagealjunction.

Oncology drugs often have significant side effects that are due tooff-target toxicity such as hematologic toxicity, neurological toxicity,cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity andencephalotoxicity. For example, taxanes such as docetaxel may cause thefollowing adverse effects: infections, neutropenia, anemia, febrileneutropenia, hypersensitivity, thrombocytopenia, myelotoxicity,myelosuppression, neuropathy, dysgeusia, dyspnea, constipation,anorexia, nail disorders, fluid retention, asthenia, pain, nausea,diarrhea, vomiting, fatigue, non-specific neuro cognitive problems,vertigo, encephalopathy, mucositis, alopecia, skin reactions andmyalgia.

Furthermore, solubilisation excipients required to formulate theoncology drugs may cause anaphylaxis, fluid retention andhypersensitivity. Premedication with corticosteroids, anti-histamines,cytokines and/or analgesics may also be required, each having their ownside effects. The macromolecules of the present invention have high drugloading, controlled-release, may passively target a particular tissueand improve solubility allowing a reduction of side effects associatedwith the oncology drug, the formulation of the drug withoutsolubilisation excipients and administration without or with reducedpremedication.

In another aspect of the invention, there is provided a method ofreducing the side effects of an oncology drug or the side-effectsrelating to the formulation of an oncology drug comprising administeringan effective amount of the macromolecule of the present invention to asubject, wherein the oncology drug is the pharmaceutically active agentof the first terminal group.

In yet another aspect of the invention, there is provided a method ofreducing hypersensitivity during chemotherapy comprising administeringan effective amount of the macromolecule of the invention to a subject.

Therapeutic regimens for cancer treatment often involve a cyclic therapywhere an oncology drug is administered once every two to four weeks.Often the drug is administered by infusion over 3 to 24 hours. In somecases to reduce the side effects of the drugs, or the risk ofhypersensitivity, especially anaphylaxis from the formulation of thedrug; premedication is required and its administration may be requiredup to 6 hours prior to treatment with the oncology drug. Such complextherapeutic regimens are time consuming and require the patient toremain in hospital from several hours to 2 days. The severe side effectsmay also limit the dose of oncology drug used and/or the number ofcycles of therapy that can be administered and therefore in some casesefficacy of the therapy is diminished.

In the present invention, the macromolecule comprising the oncology drugreduces side effects associated with the drug as it passivelyaccumulates at the tumor site or is directed to the tumor site by anappropriate targeting agent and release of the drug from the dendrimeris controlled.

The solubility of the macromolecules in aqueous solution allows them tobe formulated without harmful solubilisation excipients thereby reducingside effects of the formulation and in some cases eliminating the needfor premedication.

Furthermore, the macromolecules of the present invention need not beadministered by prolonged infusion. In some embodiments, they may beadministered by fast-infusion, for example, in less than 3 hours,including 2.5 hours, 2 hours, 1.5 hours, 1 hour or 30 minutes. In someembodiments, the macromolecule or formulation of macromolecule may beadministered as a bolus, for example, in 5 seconds to 5 minutes.

The macromolecules of the present invention may also allow the dose ofthe pharmaceutically active agent to be increased compared to thepharmaceutically active agent being administered alone. In anotheraspect of the invention there is provided a method of increasing thedose of a pharmaceutically active agent comprising administering themacromolecule of the present invention wherein the first terminal groupis the pharmaceutically active agent. In particular embodiments, themaximum tolerated dose is increased at least two fold compared to thepharmaceutically active agent when administered alone.

In particular embodiments of these aspects, the formulation of themacromolecule used in administration is substantially free ofsolubilisation excipients such as polyethoxylated caster oil (CremophorEL) and polysorbate 80.

In some embodiments where the pharmaceutically active agent istestosterone or dihydrotestosterone and the macromolecule is used in amethod of treating or preventing a disease or disorder associated withlow testosterone levels.

Low testosterone levels may result from a number of conditions. Forexample, the organs that produce testosterone (testis, ovaries) do notproduce enough testosterone (primary hypogonadism), the pituitary glandand its ability to regulate testosterone production is not workingproperly (secondary hypogonadism) or the hypothalamus may not beregulating hormone production correctly (tertiary hypogonadism).

Common causes of primary hypogonadism include undescended testicles,injury to the scrotum, cancer therapy, aging, mumps orchitis,chromosomal abnormalities, ovary conditions such as premature ovaryfailure or removal of both ovaries. Causes of secondary and tertiaryhypogonadism include damage to the pituitary gland from tumors ortreatment of nearby tumors, hypothalamus malformations such as inKellman's syndrome, compromised blood flow to the pituitary gland orhypothalamus, inflammation caused by HIV/AIDS, inflammation fromtuberculosis or sarcoides and the illegal use of anabolic steroids inbody building.

It should also be noted that obesity can also be a cause of lowtestosterone levels as obesity significantly enhances the conversion oftestosterone to oestrogen, a process that occurs predominantly in fatcells.

Symptoms of low testosterone include changes in mood (depression,fatigue, anger), decreased body hair, decreased mineral bone density(increased risk of osteoporosis), decreased lean body mass and musclestrength, decreased libido and erectile dysfunction, increased abdominalfat, rudimentary breast development in men and low or no sperm in semen.

An “effective amount” means an amount necessary at least partly toattain the desired response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of a particularcondition being treated. The amount varies depending upon the diseasebeing treated, the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated, the degree ofprotection desired, the formulation of the composition, the assessmentof the medical situation, and other relevant factors. It is expectedthat the amount will fall in a relatively broad range that can bedetermined through routine trials. An effective amount in relation to ahuman patient, for example, may lie in the range of about 0.1 ng per kgof body weight to 1 g per kg of body weight per dosage. In a particularembodiment the dosage is in the range of 1 μg to 1 g per kg of bodyweight per dosage, such as is in the range of 1 mg to 1 g per kg of bodyweight per dosage. In one embodiment, the dosage is in the range of 1 mgto 500 mg per kg of body weight per dosage. In another embodiment, thedosage is in the range of 1 mg to 250 mg per kg of body weight perdosage. In yet another embodiment, the dosage is in the range of 1 mg to100 mg per kg of body weight per dosage, such as up to 50 mg per kg ofbody weight per dosage. In yet another embodiment, the dosage is in therange of 1 μg to 1 mg per kg of body weight per dosage. Dosage regimesmay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily, weekly,monthly or other suitable time intervals, or the dose may beproportionally reduced as indicated by the exigencies of the situation.

In some embodiments the macromolecule is administered intravenously,intraarterially, intrapulmonarily, orally, by inhalation,intravesicularly, intramuscularly, intratracheally, subcutaneously,intraocularly, intrathecally or transdermally.

In some embodiments the macromolecule is administered as a bolus or byfast infusion, especially as a bolus.

In another aspect of the invention there is provided the use of amacromolecule of the invention in the manufacture of a medicament fortreating or suppressing the growth of cancer, reducing the toxicity ofan oncology drug or a formulation of an oncology drug, reducing sideeffects associated with an oncology drug or a formulation of an oncologydrug or reducing hypersensitivity upon treatment with an oncology drug;wherein the pharmaceutically active agent of the first terminal group isan oncology drug.

In yet another aspect of the invention there is provided a use of amacromolecule of the invention in the manufacture of a medicament fortreating or preventing a disease or disorder related to low testosteronelevels; wherein the pharmaceutically active agent of the first terminalgroup is testosterone.

The invention will now be described with reference to the followingExamples which illustrate some particular aspects of the presentinvention. However, it is to be understood that the particularity of thefollowing description of the invention is not to supersede thegenerality of the preceding description of the invention.

Abbreviations

Aba Acetylbutyric acid Ab Antibody Ac Acetyl ACN Acetonitrile AvStreptavadin BHAlysine Benzhydrylamide lysine Boc benzyloxycarbonyl CpOxo-cyclopentane carboxylic acid DBCO Dibenzenecyclooctyne DCCDicyclohexylcarbodiimide DCM Dichloromethane DGA Diglycolic acid DIPEAdiisopropylethylamine DMAP dimethylaminopyridine DMF DimethylformamideEtOAc Ethyl acetate DTX Docetaxel EDC1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ESI Electrosprayionisation Gem Gemcitabine Glu Glutaric acid HPLC High PerformanceLiquid Chromatography HSBA Hydrazinosulfonyl benzoic acid LCMS Liquidchromatography mass spectrometry MeOH Methanol MIDA Methyliminodiaceticacid PBS Phosphate buffered saline o-PDA Ortho-phenylenedioxydiaceticacid PDT 3,4-propylenedioxythiophene-2,5-dicarboxylic acid PEGPolyethylene glycol PSSP Dithiopropanoic acid PTX Paclitaxel PyBopBenzotriazol-1-yl-oxytripyrrolidino- phosphonium hexafluorophosphate SBSalbutamol SEC Size exclusion chromatography SRB Sulforhodamine B TDA2,2′-thiodiacetic acid TFA Trifluoroacetic acid

EXAMPLES

The dendrimers represented in the examples below include reference tothe core and the building units in the outermost generation of thedendrimer. The 1^(st) to subsurface generations are not depicted. Thedendrimer BHALys[Lys]₃₂ is representative of a 5 generation dendrimerhaving the formula BHALys[Lys]₃₂[Lys]₄[Lys]s[Lys]₁₆[Lys]₃₂, the 64surface amino groups being available to bind to terminal groups.

Preparation of the dendrimer scaffoldsBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₅₇₀]₃₂,BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[(ε-PEG₁₁₀₀]₃₂,BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-t-PEG₂₃₀₀]₃₂BHALys[Lys]₃₂[α-4-HSBA]₃₂[ε-PEG₁₁₀₀]₃₂,BHALys[Lys]₃₂[α-GILGVP-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂, andBHALys[Lys]₃₂[α-GILGVP-NH₂.TFA]₃₂[ε-t-PEG₂₃₀₀]₃₂ can be found inKaminskas et al., J Control. Release (2011) doi10.1016/j.jconrel.2011.02.005. Preparation of the dendrimer scaffolds4-azidobenzamide-PEG₁₂NEOEOEN[Su(NPN)₂][Lys]₁₆ [NH₂.TFA]₃₂ can be foundWO08/017122.

General Procedures

General Procedure A. Installation of Linkers to Drugs A

To a magnetically stirred solution of carboxylic acid linker (0.2-0.5mmol) in solvent DMF or acetonitrile (1-5 mL) at 0° C. was addedcoupling agent either EDC or DCC (1.2 equivalents). The mixture was leftto stir for 5 min., then a solution of solvent (1 mL) containing amixture of drug (0.4-1 equivalents) and DMAP (0.4-1 equivalents) wasadded dropwise. The mixture was kept at 0° C. for 1 hour then allowed towarm to ambient temperature. The volatiles were then removed in vacuoand the residue purified by preparative HPLC (BEH 300 Waters XBridgeC18, 5 μM, 30×150 mm, 40-80% ACN/water (5-40 min), no buffer) to yieldthe desired product.

General Procedure B. Installation of Linkers to Drugs B.

To a magnetically stirred solution of drug (0.3-1.0 mmol) and anhydride(2 equivalents) in DMF (3-5 mL) was added DIPEA (3 equivalents). Themixture was stirred at ambient temperature overnight. The volatiles werethen removed in vacuo and the residue purified by preparative HPLC (BEH300 Waters XBridge C18, 5 μM, 30×150 mm, 40-70% ACN/water (5-40 min), nobuffer, RT=34 min). The appropriate fractions were concentrated in vacuoproviding the desired target.

General Procedure C. Loading Dendrimer with Drug-Linker.

To a magnetically stirred mixture ofBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (0.5-1.0 μmol) and DIPEA (1.2equivalents per amine) in DMF at room temperature was added linker—drug(1.2 equivalents per amine group) and PyBOP (1.2 equivalents per aminegroup). After 1.5 hours at room temperature the volatiles were removedand the residue purified by SEC (sephadex, LH20, MeOH). The appropriatefractions, as judged by HPLC, were combined and concentrated to providethe desired material.

General Procedure D. Click Reaction

To a magnetically stirred solution dendrimer (0.5-1.0 mmol) in 1:1H₂O/t-BuOH (approximately 0.5 mL) was added alkyne reagent (2equivalents), sodium ascorbate solution (2 equivalents) and CuSO₄solution (20 mol %). The solution was heated at 80° C. and monitored byHPLC. Additional charges of both sodium ascorbate and CuSO₄ were addedas required to drive the reaction to completion. After the reaction wasjudged complete the reaction was concentrated in vacuo and thenpurified.

Example 1

(a) Preparation of 4-Aba-DTX:

Prepared using Procedure A above, using DTX (200 mg, 0.25 mmol) and4-acetylbutyric acid (42 mg, 0.32 mmol) as the linker. Preparative HPLC(RT=32 mins) provided 73 mg (32%) of product as a white solid. LCMS (C8,gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN (9-11min), 40% ACN (11-15 min), 0.1% TFA) Rt (min)=7.60. ESI (+ve) observed[M+H]⁺=920. Calculated for C₄₉H₆₁NO₁₆=919.40 Da. ¹H NMR (300 MHz, CD₃OD)δ (ppm): 1.09 (s, 3H), 1.13 (s, 3H), 1.38 (s, 9H), 1.66 (s, 3H),1.74-1.97 (m, 7H), 2.10 (s, 3H), 2.12-2.36 (m, 1H), 2.29-2.58 (m, 8H),3.83 (d, J=6.9 Hz, 1H), 4.14-4.26 (m, 3H), 4.95-5.05 (m, 2H), 5.18-5.35(m, 3H), 5.61 (d, J=7.2 Hz, 1H), 6.05 (m, 1H), 7.17-720 (m, 1H),7.23-7.45 (m, 4H), 7.52-7.62 (m, 2H), 7.63-7.72 (m, 1H), 8.10 (d, J=7.2Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-4-HSBA-4Aba-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above. To a magnetically stirred solution of4-Aba-DTX (15 mg, 16.3 μmol) in dry MeOH (1 mL) was added TFA (50 μL)and BHALys[Lys]₃₂[α-4-HSBA]₂[ε-PEG₁₁₀₀]₃₂ (20 mg, 0.43 μmol). Themixture was left to stir overnight at ambient temperature then addeddirectly to a sephadex column (LH20, MeOH) for purification. Theappropriate fractions, as judged by HPLC, were combined and concentratedto provide 25 mg (78%) of desired material as a white solid. HPLC (C8,gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11min), 40% ACN (11-15 min), 10 mM ammonium formate) Rt (min)=6.77. ¹H NMR(300 MHz, CD₃OD) δ (ppm): 0.6-2.2 (m, 812H), 2.2-2.5 (m, 115H), 2.9-3.2(m, 78H), 3.26 (s, 79H), 3.3-3.8 (m, 2824H), 5.1-5.3 (m, 31H), 5.5-5.6(m, 10H), 5.9-6.1 (m, 9H), 6.9-8.2 (m, 329H). Theoretical molecularweight of conjugate: 78.6 kDa. ¹H NMR indicates 9 DTX/dendrimer. Actualmolecular weight is approximately 56.4 kDa (13% DTX by weight).

Example 2

(a) Preparation of PSSP-DTX:

In this example (R₁═R₂═H) it could be envisioned that the rate ofrelease of docetaxel could be increased or decreased by increasing ordecreasing the degree of steric hindrance about the disulphide bond(Worrell N; R., Cumber A. J., Parnell G. D., Mirza A., Forrester J. A.,Ross W. C. J.: Effect of linkage variation on pharmacokinetics ofricin-A-chain antibody conjugates in normal rats. Anti-Cancer DrugDesign 1, 179, 1986). This could be achieved through the addition ofsubstituents, amongst others α and or β to the disulphide bond. Thistype of tuning strategy is often used in prodrug design strategies andtakes advantage of the well known Thorpe-Ingold or gem-dimethyl effect(The gem-Dimethyl Effect Revisited Steven M. Bachrach, J. Org. Chem.2008, 73, 2466-2468).

Prepared using Procedure A above, using DTX (500 mg, 0.62 mmol) and3,3′-dithiopropanoic acid (130 mg, 0.62 mmol) as the linker. PreparativeHPLC (RT=32 min) provided 179 mg (29%) of product as a white solid. LCMS(C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN(9-11 min), 40% ACN (11-15 min), 0.1% TFA) Rf (min)=7.57. ESI (+ve)observed [M+H]⁺=1000. Calculated for C₄₉H₆₁NO₁₇S₂=999.34 Da. ¹H NMR (300MHz, CD₃OD) δ (ppm): 1.13 (s, 3H), 1.17 (s, 3H), 1.43 (s, 9H), 1.70 (s,3H), 1.72-1.99 (m, 6H), 2.13-2.32 (m, 1H), 2.37-2.55 (m, 4H), 2.66-2.76(m, 2H), 2.76-3.02 (m, 6H), 3.87 (d, J=6.9 Hz, 1H), 4.18-4.31 (m, 3H),5.00-5.06 (m, 3H), 5.24-5.42 (m, 3H), 5.64 (d, J=72 Hz, 1H), 6.10 (m,1H), 7.23-7.33 (m, 1H), 7.36-7.48 (m, 4H), 7.53-7.65 (m, 2H), 7.66-7.76(m, 1H), 8.13 (d, J=7.2 Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-PSSP-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (34 mg, 0.78 μmol) and PSSP-DTX(30 mg, 30 μmol). Purification by SEC provided 50 mg (89%) of desiredmaterial as a white solid. HPLC (C8, gradient: 40-80% ACN/H₂O (1-7 min),80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mMammonium formate) Rf (min)=7.96 min. ¹H NMR (300 MHz, CD₃OD) δ (ppm):0.7-2.0 (m, 1041H), 2.0-2.2 (m, 151H), 2.2-2.5 (m, 119H), 2.5-2.7 (m,31H), 2.7-3.0 (m, 119H), 3.0-3.2 (m, 68H), 3.26 (s, 132H), 3.3-3.8 (m,2806H), 3.9-4.3 (m, 76H), 5.1-5.3 (m, 55H), 5.5-5.6 (m, 17H), 5.9-6.1(m, 17H), 7.1-8.1 (m, 243H). Theoretical molecular weight of conjugate:74.9 kDa. ¹H NMR indicates 17 DTX/dendrimer. Actual molecular weight isapproximately 56.1 kDa (24% DTX by weight).

Example 3

(a) Preparation of DGA-DTX:

Prepared using Procedure B above, using DTX (300 mg, 371 μmol) anddiglycolic anhydride (86 mg, 742 μmol) as the linker. Preparative HPLC(RT=34 min) provided 85 mg (25%) of DGA-DTX as a white solid. LCMS (C8,gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN (9-11min), 40% ACN (11-15 min), 0.1% Formic acid) Rt (min)=5.90. ESI (+ve)observed [M+H]⁺=924.10. Calculated for C₄₇H₅₇NO₁₈=923.36 Da. ¹H NMR (300MHz, CDCl₃) δ (ppm): 1.11 (s, 3H), 1.21 (s, 3H), 1.33 (s, 9H), 1.58-2.66(m, 7H), 1.73 (s, 3H), 1.93 (s, 3H), 2.67-3.67 (br s, 5H), 3.73-3.97 (brs, 1H), 4.02-4.68 (m, 7H), 4.96 (d, J=8.4 Hz, 1H), 5.24 (s, 1H),5.35-5.55 (m, 1H), 5.50 (s, 1H), 5.66 (d, J=6.7 Hz, 1H), 5.95-6.30 (m,1H), 7.24-7.68 (m, 7H), 8.08 (d, J=6.9 Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-DGA-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys][α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (36 mg, 0.84 μmol) and DOA-DTX (30mg, 33 μmol). Purification by SEC provided 45 mg (79%) of desiredmaterial as a white solid. HPLC (C8, gradient: 40-80% ACN/H₂O (1-7 min),80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mMammonium formate) Rt (min)=7.69. ¹H NMR (300 MHz, CD₃OD) δ (ppm):1.0-2.1 (m, 833H), 2.3-2.6 (m, 125H), 3.0-3.3 (m, 68H), 3.5-4.0 (m,2803H), 4.0-4.7 (m, 214H), 5.0-5.1 (m, 23H), 5.3-5.5 (m, 54H), 5.6-5.8(m, 19H), 6.0-6.3 (m, 18H), 7.2-7.8 (m, 203H), 8.1-8.2 (m, 46H).Theoretical molecular weight of conjugate: 72.4 kDa. ¹H NMR indicates 18DTX/dendrimer. Actual molecular weight is approximately 55.7 kDa (26%DTX by weight).

Example 4

(a) Preparation of Cp-DTX:

Prepared using Procedure A above, using DTX (500 mg, 619 μmol) and3-oxo-1-cyclopentanecarboxylic acid (79 mg, 619 μmol) as the linker.Preparative HPLC (RT=33.5 min) provided Cp-DTX (401 mg, 71%) as a whitesolid. LCMS (C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min),90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% Formic acid)Rt(min)=6.61. ESI (+ve) observed [M+H]J=918.54. Calculated forC₄₉H₅₉NO₁₆=917.38 Da. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 1.13 (s, 3H),1.24 (s, 3H), 1.33 (s, 9H), 1.76 (s, 3H), 1.77-2.01 (m, 3H), 1.95 (s,3H), 2.11-2.49 (m, 6H), 2.46 (s, 3H), 2.60 (ddd, J=16.2, 9.9 and 6.9 Hz,1H), 3.10-3.24 (m, 1H), 3.94 (d, J=7.2 Hz, 1H), 4.20 (d, J 8.4 Hz, 1H),4.27 (dd, J=11.1 and 6.6 Hz, 1H), 4.33 (d, J=8.4 Hz, 1H), 4.97 (d, J=7.8Hz, 1H), 5.21 (s, 1H), 5.33 (d, J=9.9 Hz, 1H), 5.42 (d, J=2.7 Hz, 1H),5.48-5.58 (br d, J=9 Hz, 1H), 5.69 (d, J=7.2 Hz, 1H), 6.27 (t, J=8.7 Hz,1H), 7.25-7.45 (m, 5H), 7.47-7.53 (m, 2H), 7.57-7.64 (m, 1H), 8.09-8.14(m, 2H).

(b) Preparation of 4-HSBA-Cp-DTX:

A solution of DTX-Cp (30 mg, 32.7 μmol) in TFA/MeOH (5% v/v, 1 mL) wasadded to 4-hydrazinosulfonylbenzoic acid (6 mg, 27.8 μmol). The mixturewas left to react at 38° C. for 1.5 h after which the solvent wasevaporated in vacuo. The white semi-solid obtained was used directly inthe next step.

(c) Preparation of BHALys[Lys]₃₂[α-4-HSBA-Cp-DTX]₃₂ [ε-PEG₁₁₀₀]₃₂

Method A:

To a magnetically stirred solution of Cp-DTX (7.5 mg, 8.15 μmol) in dryMeOH (1 mL) was added TFA (50 μL). This solution was added toBHALys[Lys]₃₂ [α-4-HSBA]₃₂[ε-PEG₁₁₀₀]₃₂ (10 mg, 0.215 μmol). The mixturewas left to react overnight at ambient temperature then added directlyto a sephadex column (LH20, MeOH) for purification. The appropriatefractions, as judged by HPLC, were combined, concentrated andfreeze-dried from water to provide 18 mg (70%) of desired material as awhite solid.

Method B:

To 4-HSBA-Cp-DTX (31 mg, 27.8 μmol) and PyBOP (14.5 mg, 27.8 μmol) wasadded a solution of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (31.5 mg,0.7 μmol) and DIPEA (15 μL, 89.0 μmol) in DMF (1 mL). The resultingmixture was stirred overnight at ambient temperature after which thesolvent was evaporated in vacuo. The remaining yellow oil was added to asephadex column (LH20, MeOH) for purification. The appropriatefractions, as judged by HPLC, were combined, concentrated andfreeze-dried from water to provide 34 mg (81% over two steps) of desiredmaterial as a white solid. HPLC (C8, gradient: 40-80% ACN/H₂O (1-7 min),80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mMammonium formate) Rt (min)=7.65. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.12(s, 44H), 1.16 (s, 44H), 1.21-2.29 (m, 688H), 2.32-2.53 (m, 113H),2.80-3.25 (m, 64H), 3.35 (s, 85H), 3.36-3.90 (m, 2815H), 4.17-4.28 (m,77H), 4.45-4.65 (m, 50H), 4.97-5.04 (m, 23H), 5.22-5.44 (m, 40H), 5.63(d, J=6.9 Hz, 16H), 6.00-6.20 (m, 15H), 7.2-8.25 (m, 308H). Theoreticalmolecular weight of conjugate: 78.8 kDa. ¹H NMR indicates 15DTX/dendrimer in each case. Actual molecular weight is approximately60.0 kDa (20% DTX by weight).

Example 5

(a) Preparation of Glu-DTX:

Prepared using Procedure B above, using DTX (300 mg, 371 μmol) andglutaric anhydride (85 mg, 742 μmol) in DMF (3.7 mL) as the linker.Preparative HPLC (Rt=33 min) provided 106 mg (31%) of Glu-DTX as a whitesolid. LCMS (C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min),90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% Formic acid) Rt(min)=6.12. ESI (+ve) observed [M+H]⁺=922.13. Calculated forC₄₈H₅₉NO₁₇=921.38 Da. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 1.11 (s, 3H),1.22 (s, 3H), 1.33 (s, 9H), 1.74 (s, 3H), 1.79-2.65 (m, 14H), 1.93 (s,3H), 3.91 (d, J=6.5 Hz, 1H), 4.19 (d, J=8.4 Hz, 1H), 4.26 (dd, J=11.1and 6.9 Hz, 1H), 4.31 (d, J- 8.4 Hz, 1H), 4.96 (d, J=8.2 Hz, 1H), 5.23(s, 1H), 5.38 (br s, 1H), 5.35-5.65 (br d, 1H), 5.67 (d, J=6.5 Hz, 1H),6.10-6.30 (s, 1H), 7.26-7.34 (m, 3H), 7.34-7.43 (m, 2H), 7.46-7.55 (m,2H), 7.57-7.65 (m, 1H), 8.10 (d, J=7.4 Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-Glu-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (50 mg, 1.1 μmol) and Glu-DTX(39 mg, 42.3 μmol). Purification by sephadex column (LH20, MeOH)provided 49.5 mg (78%) of desired material as a white solid. HPLC (C8,gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11min), 40% ACN (11-15 min), 10 mM ammonium formate) Rt (min)=7.78. ¹H NMR(300 MHz, CD₃OD) δ (ppm): 1.00-2.10 (m, 1037H), 2.10-2.74 (m, 296H),3.05-3.27 (br s, 88H), 3.35 (s, 96H), 3.36-3.78 (m, 2800H), 3.80-3.93(m, 42H), 4.01-4.47 (m, 125H), 4.47-4.60 (br a, 23H), 4.92-5.08 (br s,30H), 5.18-5.45 (m, 70H), 5.54-5.74 (br s, 22H), 6.00-6.23 (br s, 20H),7.15-7.75 (m, 414H), 8.05-8.20 (br d, J=6.4 Hz, 49H). Theoreticalmolecular weight of conjugate: 72.6 kDa. ¹H NMR indicates 20DTX/dendrimer. Actual molecular weight is approximately 57.5 kDa (28%DTX by weight).

Example 6

(a) Preparation of MIDA-DTX:

Prepared using Procedure A above, using DTX (100 mg, 124 μmol) andmethyliminodiacetic acid (91 mg, 620 μmol) as the linker. PreparativeHPLC (RT=22.5 min) provided 29 mg (25%) of product as a white solid.LCMS (C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40%ACN (9-11 min), 40% ACN (11-15 min), 0.1% Formic acid) Rt (min)=4.62.ESI (+ve) observed [M+H]⁺=937.34. Calculated for C₄₈H₆₀N₂O₁₇=936.39 Da.¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.13 (s, 3H), 1.17 (s, 3H), 1.40 (s,9H), 1.70 (s, 3H), 1.84 (ddd, J=14.1, 11.4 and 1.8 Hz, 1H), 1.93 (s,3H), 2.04 (dd, J=15.0 and 8.7 Hz, 1H), 2.30 (dd, J=15.0 and 8.7 Hz, 1H),2.43 (s, 3H), 2.46 (ddd, J=14.1, 9.5 and 6.6 Hz, 1H), 2.61 (s, 3H), 3.49(s, 2H), 3.81-3.94 (m, 3H), 4.21 (s, 2H), 4.24 (dd, J=11.4 and 6.6 Hz,1H), 5.01 (dd, J=9.5 and 1.8 Hz, 1H), 5.29 (s, 1H), 5.43 (s, 2H), 5.65(d, J=7.2 Hz, 1H), 6.16 (t, J=8.7 Hz, 1H), 7.21-7.34 (m, 1H), 7.35-7.50(m, 4H), 7.51-7.79 (m, 3H), 8.13 (d, J=7.2 Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-MIDA-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys][α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (31.5 mg, 0.7 μmol) and MIDA-DTX(26 mg, 27.8 μmol). Purification by SEC provided 41.6 mg (93%) of thedesired product as a white solid. HPLC (C8, gradient: 40-80% ACN/H₂O(1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40%6 ACN (11-15min), 10 mM ammonium formate) Rt (min)=7.78. ¹H NMR (300 MHz, CD₃OD) δ(ppm): 1.00-2.10 (m, 1186H), 2.12-2.68 (m, 283H), 3.06-3.27 (m, 77H),3.35 (s, 101H), 3.36-3.96 (m, 2842H), 4.07-4.61 (m, 143H), 4.93-5.10 (brs, 31H), 5.19-5.48 (m, 77H), 5.55-5.75 (m, 27H), 5.97-6.29 (m, 27H),7.10-7.84 (m, 258H), 8.03-8.23 (m, 60H). Theoretical molecular weight ofconjugate: 73.1 kDa. ¹H NMR indicates 27 DTX/dendrimer. Actual molecularweight is approximately 64.2 kDa (34% DTX by weight).

Example 7

(a) Preparation of o-PDA-DTX

Prepared using Procedure A above, using DTX (300 mg, 0.37 mmol) ando-phenylenedioxydiacetic acid (419 mg, 1.85 mmol) as the linker.Preparative HPLC (RT=26 min) provided 21 mg (11%) of product as a whitesolid. LCMS (C8, gradient: 40-90%, ACN/H₂O (1-7 min), 90% ACN (7-9 min),90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% Formic acid) Rt (min)7.27. ESI (+ve) observed [M+H]⁺=1016.29. Calculated forC₅₃H₆₁NO₁₉=1015.38 Da. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.13 (s, 3H),1.17 (s, 3H), 1.40 (s, 9H), 1.69 (s, 3H), 1.82 (ddd, J=13.5, 11.4 and2.1 Hz, 1H), 1.89 (s, 3H), 1.94-2.07 (m, 1H), 2.00-2.33 (m, 1H), 2.40(s, 3H), 2.45 (ddd, J=15.9, 9.6 and 6.6 Hz, 1H), 3.87 (d, J=6.9 Hz, 1H),4.18-4.27 (m, 3H), 4.68 (s, 2H), 4.87 (d, J=6.0 Hz, 1H), 5.00 (d, J=9.3Hz, 1H), 5.27 (s, 1H), 5.36-5.43 (m, 2H), 5.64 (d, J=6.9 Hz, 1H), 6.13(t, J=9.0 Hz, 1H), 6.86-6.98 (m, 4H), 7.23-7.32 (m, 1H), 7.35-7.43 (m,4H), 7.52-7.60 (m, 2H), 7.62-7.70 (m, 1H), 8.07-8.15 (m, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-o-PDA-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (22.5 mg, 0.5 μmol) ando-PDA-DTX (21 mg, 20.7 μmol). Purification by SEC (sephadex, LH20, MeOH)provided 30 mg (95%) of the desired product as a slightly beigesemi-solid. HPLC (C8, gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9min), 80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mM ammoniumformate) Rt (min)=9.80. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.95-2.12 (m,1058H), 2.12-2.66 (m, 205H), 2.89-3.29 (m, 125H), 3.35 (s, 85H),3.36-3.93 (m, 2822H), 3.98-4.75 (m, 212H), 4.83-5.08 (m, 89H), 5.18-5.34(m, 17H), 5.34-5.54 (m, 38H), 5.54-5.79 (m, 22H), 6.01-6.26 (m, 22H),6.68-7.13 (m, 98H), 7.13-7.78 (m, 214H), 8.02-8.22 (m, 50H). Theoreticalmolecular weight of conjugate: 75.6 kDa. ¹H NMR indicates 22DTX/dendrimer. Actual molecular weight is approximately 63.2 kDa (28%4DTX by weight).

Example 8

(a) Preparation of TDA-DTX Via Procedure A:

Prepared using Procedure A above, using DTX (500 mg, 0.62 mmol) and2,2′-thiodiacetic acid (370 mg, 2.5 mmol) as the linker. PreparativeHPLC (RT=33 min) provided 240 mg (41%) of product as a white solid. LCMS(C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN(9-11 min), 40% ACN (11-15 min), 0.1% TFA) Rt (min)=10.60. ESI (+ve)observed [M+H]⁺=940. Calculated for C₄₇H₅₇NO₁₇S=939.33 Da. ¹H NMR (300MHz, CD₃OD) δ (ppm): 1.15 (s, 3H), 1.19 (s, 31H), 1.43 (s, 9H), 1.72 (s,3H), 1.78-2.05 (m, 2H), 1.93 (s, 3H), 2.16-2.57 (m, 2H), 2.43 (s, 3H),336-3.63 (m, 2H), 3.89 (d, J=6.9 Hz, 1H), 4.18-4.34 (m, 3H), 5.03 (d,J=9.0 Hz, 2H), 5.28-5.44 (m, 3H), 5.66 (d, J=7.2 Hz, 1H), 6.11 (m, 1H),7.24-7.35 (m, 1H), 7.38-7.50 (m, 4H), 7.52-7.65 (m, 2H), 7.66-7.76 (m,1H), 8.14 (d, J=7.2 Hz, 2H).

(b) Preparation of TDA-DTX Via Procedure B:

Prepared using Procedure B above, using DTX (400 mg, 0.50 mmol) andthiodiacetic anhydride (66 mg, 0.50 mmol) as the linker. The mixture wasstirred at room temperature overnight then solvent was removed underreduced pressure to give a crude residue. The residue was re-dissolvedin EtOAc (250 mL) and was washed with PBS buffer (adjusted to pH 4.0).The separated organic layer was dried over MgSO₄ and concentrated underreduced pressure to give 445 mg (95%) of the desired product as a whitesolid. LCMS (Waters XBridge C8 column (3.0×100 mm), 3.5 micron, 214, 243an, 0.4 mL/min, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min),90-40% ACN (9-11 min), 40% ACN 11-15 min), 0.1% TFA) Rt (min)=10.60. ESI(+ve) observed [M+H]+=940. Calculated for C₄₇H₅₇NO₁₇S=939.33 Da.

(c) Preparation of BHALys[Lys]₃₂[α-TDA-DTX]₃₂[α-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA][ε-PEG₁₁₀₀]₃₂ (46 mg, 1.08 μmol) and TDA-DTX (44mg, 47 μmol). Purification by SEC (sephadex, LH20, MeOH) provided 65 mg(87%) of desired material as a white solid. HPLC (C8, gradient: 40-80%ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN(11-15 min), 10 mM ammonium formate) Rt (min)=9.68. ¹H NMR (300 MHz,CD₃OD) δ (ppm): 0.78-2.02 (m, 809H), 2.27-2.58 (m, 114H), 3.03-3.24 (m,43H), 3.34 (s, 73H), 3.37-3.96 (m, 2800H), 4.01-4.39 (m, 27H), 5.20-5.48(m, 75H), 5.54-5.74 (m, 23H), 5.98-6.25 (m, 20H), 7.12-7.84 (m, 202H),8.01-8.22 (m, 46H). Theoretical molecular weight of conjugate: 68.9 kDa.¹H NMR indicates 23 DTX/dendrimer. Actual molecular weight isapproximately 60.6 kDa (31% DTX by weight). Particle sizing usingDynamic Light Scattering shows a range of concentration dependentaverages of 8.9-10.1 nm.

Example 9

(a) Preparation of PDT-DTX:

Prepared using Procedure A above, using DTX (250 mg, 0.31 mmol) and3,4-propylenedioxythiophene-2,5-dicarboxylic acid (PDT, 75 mg, 0.31mmol) as the linker. Purification by preparative HPLC (RT=28 min)provided 30 mg (9%) of product as a white solid. LCMS (C8, gradient:40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN (9-11 min), 40%ACN (11-15 min), 0.1% TFA) Rt (min)=7.24. ESI (+ve) observed[M+H]⁺⁼1034. Calculated for C₅₂H₅₉NO₁₉S=1033.34 Da. ¹H NMR (300 MHz,CD₃OD) δ (ppm): 1.14 (s, 3H), 1.18 (s, 3H), 1.45 (s, 9H), 1.71 (s, 3H),1.78-1.91 (m, 2H), 1.94 (s, 3H), 2.09-2.27 (m, 1H), 2.29-2.58 (m, 3H),2.41 (s, 3H), 3.88 (d, J=6.9 Hz, 1H), 4.20-4.30 (m, 3H), 4.31-4.43 (m,4H), 4.94-5.16 (m, 1H), 5.30 (s, 1H), 5.36-5.42 (m, 2H), 5.65 (d, J=6.9Hz, 1H), 6.02-6.22 (m, 1H), 7.23-7.34 (m, 1H), 7.36-7.53 (m, 4H),7.56-7.65 (m, 2H), 7.66-7.77 (m, 1H), 8.11 (d, J=7.2 Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-PDT-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA][ε-PEG₁₁₀₀]₃₂ (29 mg, 0.67 μmol) and PDT-DTX (30mg, 29 μmol). Purification by SEC (sephadex, LH20, MeOH) provided 42 mg(88%) of desired material as a white solid. HPLC (C8, gradient: 40-80%ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN(11-15 min), 10 mM ammonium formate) Rt (min)=9.03. ¹H NMR (300 MHz,CD₃OD) δ (ppm): 0.76-2.10 (m, 974H), 2.23-2.66 (m, 210H), 3.08-3.30 (m,74H), 3.40-3.98 (m, 2804H), 4.02-4.76 (m, 249H), 4.96-5.12 (m, 33H),5.22-5.34 (m, 25H), 5.36-5.52 (m, 47H), 5.56-5.80 (m, 27H), 5.88-6.30(m, 24H), 7.08-7.94 (m, 213H), 7.99-8.31 (m, 50H). Theoretical molecularweight of conjugate: 71.9 kDa. ¹H NMR indicates 26 DTX/dendrimer. Actualmolecular weight is approximately 66.3 kDa (32% DTX by weight).

Example 10

(a) Preparation of PEG₂-DTX:

Prepared using Procedure A above, using DTX (200 mg, 0.25 mmol) and3,6,9-trioxaundecanedioic acid (220 mg, 1.0 mmol). Preparative HPLC(RT=30.5 min) provided 70 mg (28%) of product as a white solid. LCMS(C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN (7-9 min), 90-40% ACN(9-11 min), 40% ACN (11-15 min), 0.1% Formic acid) Rt (min)=6.48. ESI(+ve) observed [M+H]⁺=1012.15. Calculated for C₅₁H₆₅NO₂₀=1011.41 Da. ¹HNMR (300 MHz, CD₃OD) δ (ppm): 1.13 (s, 3H), 1.17 (s, 3H), 1.40 (s, 9H),1.70 (s, 3H), 1.83 (ddd, J=13.8, 11.1 and 2.1 Hz, 1H), 1.93 (s, 3H),1.92-2.12 (m, 1H), 2.17-2.38 (m, 1H), 2.42 (s, 3H), 2.46 (ddd, J=14.7,9.9 and 6.6 Hz, 1H), 3.56-3.82 (m, 8H), 3.88 (d, J=7.0 Hz, 1H), 4.06 (s,2H), 4.16-4.39 (m, 5H), 5.01 (d, J=9.3 Hz, 1H), 5.29 (s, 1H), 5.38 (s,2H), 5.65 (d, J=7.0 Hz, 1H), 6.13 (t, J=8.4 Hz, 1H), 7.22-7.33 (m, 1H),7.35-7.47 (m, 4H), 7.51-7.62 (m, 2H); 7.62-7.72 (m, 1H), 8.13 (d, J=7.2Hz, 2H).

(b) Preparation of BHALys[Lys]₃₂[α-PEG₂-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA][ε-PEG₁₁₀₀]₃₂ (55.8 mg, 1.24 μmol) and PEG₂-DTX(50 mg, 49.5 μmol). Purification by SEC (sephadex, LH20, MeOH) provided79 mg (>90%) of the desired product as a white solid. HPLC (C8,gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11min), 40% ACN (11-15 min), 10 mM ammonium formate) Rf (min)=8.65. ¹H NMR(300 MHz, CD₃OD) δ (ppm): 0.91-2.14 (m, 968H), 2.14-2.64 (m, 185H),2.88-3.29 (m, 109H), 3.35 (s, 89H), 3.36-3.95 (m, 3016H), 3.95-4.65 (m,251H), 5.00 (br s, 32H); 5.20-5.49 (m, 72H), 5.55-5.75 (m, 25H), 6.13(br s, 25H), 7.12-7.81 (m, 213H), 8.13 (d, J=7.2 Hz, 50H). Theoreticalmolecular weight of conjugate: 75.5 kDa. ¹H NMR indicates 24DTX/dendrimer. Actual molecular weight is approximately 63.2 kDa (31%DTX by weight).

Example 11

Preparation of BHALys[Lys]₃₂[α-Lys(α-Ac)(ε-DGA-DTX)]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

(a) Preparation of HO-Lys(NH₂.TFA)₂

To a magnetically stirred suspension of L-lysine (500 mg, 3.42 mmol) inCH₂Cl₂ (21 mL) was added a solution of TFA in CH₂Cl₂ (21 mL, 1:1 v/v).The mixture was stirred at ambient temperature for 4 h, and thenconcentrated in vacuo. The residue was dissolved in water (30 mL) andconcentrated in vacuo. This procedure was repeated once more. Theremaining oil was then freeze-dried from water, providing 1.33 g of thedesired product as a yellowish oil that was used directly in the nextstep.

(b) Preparation of HO-Lys(PEG₅₇₀)₂

To a magnetically stirred solution of PEG₅₇₀-NHS (1.06 g, 1.55 mmol) inDMF (5 mL) was added DIPEA (806 μL, 4.64 mmol), followed by a solutionof HO-Lys(NH₂.TFA)₂ (300 mg) in DMF (4 mL). The resulting mixture wasstirred at ambient temperature overnight. The volatiles were thenremoved in vacuo and the residue purified by preparative HPLC (BEH 300Waters XBridge C18, 5 μM, 30×150 mm, gradient: 5% ACN/H₂O (1-5 min),5-60% ACN (5-35 min), 60-80% ACN (35-40 min), 80% ACN (40-45 min), 80-5%ACN (45-50 min), 5% ACN (50-60 min), no buffer, Rt=29.3 min). Theappropriate fractions were concentrated in vacuo and freeze-dried inwater, providing 481 mg (48% over two steps) of the desired product as awhite semi-solid. HPLC (C18, gradient: 5-60% ACN/H₂O (1-10 min), 60% ACN(10-11 min), 60-5% ACN (11-13 min), 5% ACN (13-15 min), 10 mM ammoniumformate) Rt (min)=8.68. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.33-1.62 (m,4H), 1.62-1.95 (m, 2H), 2.43 (t, J=6.2 Hz, 2H), 2.52 (dt, J=6.2 and 3.6Hz, 2H), 3.16-3.24 (m, 2H), 3.36 (s, 6H), 3.36-3.90 (m, 95H), 4.39 (dd,J=8.7 and 5.1 Hz, 1H).

(c) Preparation of BHALys[Lys]₁₆[Lys(α-Boc)(ε-NH₂]₃₂

To a magnetically stirred suspension ofBHALys[Lys]₁₆[Lys(α-Boc)(ε-Fmoc)]₃₂ (500 mg, 26.9 μmol) in DMF (3.4 mL)was added piperidine (849 μL, 20% v/v in DMF). The mixture was stirredat ambient temperature overnight, then poured into diethyl ether (65mL). The white precipitate that formed was filtered off and washed withdiethyl ether (100 mL). The filter cake was transferred to a vial andair dried for 3 days, providing 281 mg (91%) product as a white solid.¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.00-2.10 (m, 680H), 2.65-2.88 (br s,48H), 2.91-2.98 (m, 11H), 2.99-3.28 (m, 78H), 3.81-4.21 (m, 33H),4.21-4.55 (m, 32H), 6.21 (s, 1H), 7.20-7.41 (m, 10H).

(d) Preparation of BHALys[Lys]₃₂[α-Boc]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

To a magnetically stirred solution of BHALys[Lys]₁₆[Lys(α-Boc)(ε-NH₂)]₃₂(49 mg, 4.33 μmol) in DMF and DMSO (3 mL, 5:1 v/v) was added DIPEA (96μL, 554.2 μmol). The resulting solution was added to a solution ofHO-Lys(PEG₅₇₀)₂ (223 mg, 173.3 μmol) and PyBOP (90 mg, 173.3 μmol) inDMF (5.5 mL). The mixture was stirred at ambient temperature overnight.The volatiles were then removed in vacuo and the residue purified byultrafiltration (Pall Minimate™ Tangential Flow Filtration Capsules,Omega™ 10K Membrane, water). The remaining aqueous solution wasfreeze-dried, providing 120 mg (53%) of the desired product as ayellowish oil. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.18-1.98 (m, 863H),2.38-2.63 (m, 123H), 3.04-3.30 (m, 194H), 3.36 (s, 172H), 3.38-3.91 (m,2816H), 3.93-4.18 (br s, 35H), 4.18-4.47 (m, 63H), 4.47-4.60 (m, 12H),6.18 (s, 1H), 7.19-7.43 (m, 10H).

(e) Preparation of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

To a magnetically stirred solution ofBHALys[Lys]₃₂[α-Boc]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂ (120 mg, 2.3 μmol) in CH₂Cl₂ (2mL) was added a solution of TFA in CH₂Cl₂ (2 mL, 1:1 v/v). The mixturewas stirred at ambient temperature for 3.5 h, after which the solventswere evaporated in vacuo. The remaining oil was dissolved in water (5mL) and the resulting solution concentrated in vacuo. This procedure wasrepeated one more time and the oil that remained was taken up in waterand purified by SEC (PD-10 desalting columns, GE Healthcare, 17-0851-01,sephadex 0-25 medium). The collected fractions were combined andfreeze-dried from water to provide 93 mg (77%) of desired material as ayellowish oil. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.18-2.01 (m, 556H),2.38-2.65 (m, 118H), 3.02-3.30 (m, 181H), 3.36 (s, 178H), 3.38-3.94 (m,2816H), 4.09-4.55 (m, 63H), 6.13-6.22 (m, 1H), 7.19-7.45 (m, 10H).

(f) Preparation of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

To a solution of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂ (93 mg,1.8 μmol) in DMF (3.6 mL) was added DIPEA (40 μL, 230.4 μmol). Theresulting solution was added to solid HO-Lys(α-Ac)(ε-Boc) (21 mg, 72μmol) and PyBOP (37 mg, 72 μmol) contained in a second flask. Themixture was stirred at ambient temperature overnight. The volatiles werethen removed in vacuo and the residue purified by SEC (sephadex, LH20,MeOH). The appropriate fractions, as judged by HPLC were combined andconcentrated. The yellowish oil thus obtained was freeze dried fromwater to give 97 mg (94%) of the desired product as a slightly yellowishsemi-solid. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.10-2.15 (m, 1139H),2.36-2.63 (m, 120H), 2.93-3.30 (m, 251H), 3.36 (s, 195H), 3.37-3.91 (m,2816H), 4.16-4.51 (br s, 122H), 6.15-6.21 (m, 1H), 7.18-7.43 (m, 10H).

(g) Preparation ofBHALys[Lys]₃₂[α-Lys(α-Ac)(ε-NH₂.TFA]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

To a magnetically stirred solution ofBHALys[Lys]₃₂[α-Lys(α-Ac)(ε-Boc)]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂ (97 mg, 1.69 μmol)in CH₂Cl₂ (1 mL) was added a solution of TFA in CH₂Cl₂ (2 mL, 1:1 v/v).The mixture was stirred at ambient temperature overnight, and then thesolvents were evaporated in vacuo. The remaining oil was dissolved inwater (4 mL) and the resulting solution concentrated in vacuo. Thisprocedure was repeated one more time and the oil that remained was takenup in water and purified by SEC (PD-10 desalting columns, GE Healthcare,17-0851-01, sephadex 0-25 medium). The collected fractions were combinedand freeze-dried from water to provide 104 mg (>90%) of the desiredmaterial as a yellowish oil. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 1.13-2.20(m, 843H), 2.37-2.65 (m, 122H), 2.89-3.06 (m, 70H), 3.06-3.30 (m, 180H),3.36 (s, 182H), 3.39-3.92 (m, 2816H), 4.08-4.47 (br s, 126H), 6.13-6.20(m, 1H), 7.20-7.45 (m, 10H).

(k) Preparation ofBHALys[Lys]₃₂[α-Lys(α-Ac)(εDGA-DTX)]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-Lys(α-Ac)(ε-NH₂.TFA]₃₂[ε-Lys(PEG₅₇₀)₂]₃₂ (49 mg, 0.85μmol) and DOA-DTX (31 mg, 34 μmol). Purification by SEC (sephadex, LH20,MeOH) provided 57 mg (80%) of the desired product as a white solid. HPLC(C8, gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN(9-11 min), 40% ACN (11-15 min), 10 mM ammonium formate) Rt (min)=8.85.¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.79-2.73 (m, 1698H), 3.06-3.29 (m,179H), 3.35 (s, 184H), 3.36-3.92 (m, 2848H), 3.95-4.60 (m, 332H), 5.01(br s, 32H), 5.20-5.52 (m, 77H), 5.64 (br s, 30H), 6.13 (br s, 27H),7.14-7.34 (m, 39H), 7.34-7.52 (m, 104H), 7.52-7.76 (m, 87H), 8.02-8.24(m, 57H). Theoretical molecular weight of conjugate: 83.3 kDa. ¹H NMRindicates 27 DTX/dendrimer. Actual molecular weight is approximately78.8 kDa (28% DTX by weight).

Example 12

Preparation of BHALys[Lys]₃₂[α-Glu-PTX]₃₂[ε-PEG₂₃₀₀]₃₂ PTX=Paclitaxel

Prepared using Procedure C above, using Glu-PTX (300 mg, 371 mol) andBHALys[Lys]₁₆[Lys(α-NH₂.TFA)(ε-PEG₂₃₀₀)]₃₂ (22.0 mg, 0.26 μmol).Purification by preparative HPLC (Rt=28 min) provided 12 mg (41%) of thedesired dendrimer. ¹H NMR (CD₃OD): δ 0.78-2.80 (m, 1785H), 2.96-3.23 (m,120H), 3.35-3.45 (m, 567H), 3.46-3.94 (m, 5610H), 4.04-4.47 (m, 167H),4.48-4.65 (m, 88H), 5.50 (m, 29H), 5.64 (m, 24H), 5.85 (m, 27H), 6.10(m, 26H), 6.46 (m, 20H), 7.26 (m, 66H), 7.36-8.00 (m, 407H), 8.12 (s,53H). Theoretical molecular weight of conjugate: 112.4 kDa. ¹H NMRindicates 25 PTX/dendrimer. Actual molecular weight is approximately 105kDa (20% PTX by weight).

Example 13

Preparation of BHALys[Lys]₃₂[α-Glu-GEM]₃₂[ε-PEG₁₁₀₀]₃₂ GEM=gemcitabine

(a) Preparation of N,O-di-BOC-GEM-Glu

To a stirred mixture of N,O-diBoc gemicitabine (Guo, Z.; Gallo, J. M.Selective Protection of 2′,2′Difluorodeoxycytidine J. Org. Chem, 1999,64, 8319-8322) (200 mg, 0.43 mmol) in DMF (2 mL) at 0° C. was addedDIPEA (0.4 mL, 2.15 mmol) and glutaric anhydride (100 mg, 0.86 mmol).The reaction was allowed to warm up to ambient temperature over 1 hour,then stirred for a further 3 hours. The DMF was then removed in vacuoand residue was taken up in ethyl acetate (20 mL). This mixture was thenwashed with NaHCO₃ (10%, 2×10 mL), water (2×20 mL) and brine (20 mL).The organic phase was then dried (Na₂SO₄), filtered and concentratedunder reduced pressure. The crude was then purified by silica gelchromatography (DCM/Methanol) providing 130 mg (54%) of the desiredproduct as a white solid. LCMS (C18, gradient: 20-60% ACN/H₂O (1-7 min),60% ACN (7-9 min), 60-20% ACN (9-11 min), 20% ACN (11-15 min), 0.1% TFA,Rt (min)=10.8 min. ESI (+ve) observed [M+H]=578. Calculated forC₂₄H₃₂N₃F₂O₁₁=576.20 Da. ¹H NMR (CDCl₃): δ 1.51 (s, 18H), 2.01-1.88 (m,2H), 2.55-2.4 (m, 2H), 2.75-2.64 (m, 2H), 4.46-4.38 (m, 3H), 5.15-5.10(m, 1H), 6.46-6.30 (m, 1H), 736-7.50 (d, J=7.8 Hz, 1H), 7.6-7.79 (d,J=7.8 Hz, 1H).

(b) Preparation of BHALys[Lys]₃₂[α-Glu-GEM]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₁₆[Lys(α-NH₂.TFA)(ε-PEG)₁₁₀₀]₃₂ (40 mg, 1.03 mmol) andN,O-di-Boc-GEM-Glu (28 mg, 49 μmol). Purification by SEC (PD-10desalting column, GE Healthcare, 17-0851-01, sephadex G-25 medium)provided 20 mg of material. The solid was taken up in TFA/DCM (1:1, 2mLs) and stirred for 3 hours at room temperature. The volatiles wereremoved in vacuo and the residue taken up in water and freeze dried,providing 18 mg (47%) of white powder. HPLC (C8, gradient: 40-80%ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN(11-15 min), 0.1% TFA), Rt (min)=6.06. ¹H NMR (CD₃OD): δ 0.89-2.1 (m,456H), 2.1-2.7 (m, 185H), 2.9-3.2 (m, 90H), 3.2-3.3 (m, 191H), 3.44-4.12(m, 2650H), 4.14-4.70 (m, 160H), 5.8-6.0 (m, 28H), 6.2-6.4 (m, 28H),7.05-7.15 (s, 11H), 7.5-7.7 (m, 24H). Theoretical molecular weight ofconjugate: 59.2 kDa. ¹H NMR indicates 26 GEM/dendrimer. Actual molecularweight is approximately 52.3 kDa (15% GEM by weight).

Example 14

(a) Preparation of BHALys[Lys]₃₂[αGGG-Boc]₃₂[ε-PEG₁₁₀₀]₃₂

To a magnetically stirred solution of Boc-GGG-OH (28 mg, 93.2 μmol) andPyBOP (48 mg, 93.2 μmol) in DMF (1 mL) at room temperature was added asolution of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (100 mg, 2.33 μmol)and DIPEA (51 μL, 298.24 μmol) in DMF (2.6 mL). The mixture was stirredat room temperature for 18 b and then concentrated under reducedpressure. The residue was dissolved in MeOH (1 mL) and purified by SEC(Sephadex, LH-20, MeOH). The appropriate fractions, as judged by HPLC,were combined and concentrated to provide 98 mg of product as a clear,colourless oil. The latter was dissolved in MQ water and lyophilised togive 98 mg (87%) of product as a colourless resin. LCMS (C8, gradient:5-80% ACN/H₂O (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5%ACN (13-15 min), 0.1% TFA) Rt (min)=8.63. ¹H NMR (300 MHz, CD₃OD) δ(ppm): 1.15-2.01 (m, 693H), 2.46 (br s, 57H), 3.18 (br s, 101H), 3.35(s, 53H), 3.36 (s, 84H), 3.38-4.04 (m, 2990H), 4.30 (br s, 63H), 6.17(br s, 1H), 7.29 (br s, 9H). ¹H NMR indicates ca. 32 Boc-GGG/dendrimer.Molecular weight is approximately 48.5 kDa.

(b) Preparation of BHALys[Lys]₃₂[α-GGG-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂

To a magnetically stirred mixture ofBHALys[Lys]₃₂[α-GGG-Boc]₃₂[ε-PEG₁₁₀₀]₃₂ (98 mg, 2.02 μmol) in CH₂Cl₂ (1mL) at room temperature was added a solution of TFA in CH₂Cl₂ (1:1, 2mL). After 18 hours at room temperature the volatiles were removed. Theresulting residue was dissolved in MQ water (15 mL) and concentrated.This procedure was repeated once more. The residue was then dissolved inMQ water (12.5 mL) and purified by SEC (PD-10, MQ water). Theappropriate fractions were combined and lyophilised to provide 92 mg(94%) of desired material as a clear, colourless oil. HPLC (C8,gradient: 5-80% s ACN/H₂O (1-7 min), 80% ACN (7-12 min), 80-5% ACN(12-13 min), 5% ACN (13-15 min), 0.1% TFA) Rt (min)=7.94. ¹H NMR (300MHz, CD₃OD) δ (ppm): 1.19-2.05 (m, 351H), 2.47 (br s, 58H), 3.18 (br s,105H), 3.36 (s, 89H), 3.38-4.15 (m, 2990H), 4.31 (br s, 72H), 6.17 (brs, 1H), 7.30 (br s, 9H). ¹H NMR indicates ca. 32 GGG-NH₂.TFA/dendrimer.Molecular weight is approximately 48.6 kDa.

(c) Preparation of BHALys[Lys]₃₂[α-GGG-Glu-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[(α-GGG-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (75 mg, 1.53 μmol) andGlu-DTX (56 mg, 61.2 μmol). Purification by SEC (Sephadex, LH-20, MeOH)provided 96 mg (92%) of product as a white solid. HPLC (C8, gradient:5-80% ACN/H₂O (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5%ACN (13-15 min), 0.1% TFA) Rt (min)=10.08. ¹H NMR (300 MHz, CD₃OD) δ(ppm): 0.75-2.02 (m, 985H), 2.02-2.64 (m, 309H), 2.92-3.17 (m, 53H),3.25 (s, 89H), 3.26-4.00 (m, 3070H), 4.00-4.40 (m, 174H), 4.82-5.00 (m,44H), 5.04-5.39 (m, 87H), 5.54 (br s, 27H), 6.01 (br s, 22H), 7.03-7.67(m, 227H), 7.92-8.10 (m, 49H). Theoretical molecular weight ofconjugate: 73.9 kDa. ¹H NMR indicates 32 GGG and 26 DTX/dendrimer.Actual molecular weight is approximately 68.5 kDa (31% DTX by weight).

Example 15

(a) Preparation of BHALys[Lys]₃₂[α-GFLG-Boc]₃₂[ε-PEG₁₁₀₀]₃₂

To a magnetically stirred solution of Boc-GLFG-OH (32 mg, 65.2 μmol) andPyBOP (34 mg, 65.2 μmol) in DMF (1 mL) at room temperature was added asolution of BHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (70 mg, 1.63 μmol)and DIPEA (36 μL, 208.64 μmol) in DMF (1.5 mL). The mixture was stirredat room temperature for 18 h and then concentrated under reducedpressure. The residue was dissolved in MeOH (1 mL) and purified by SEC(Sephadex, LH-20, MeOH). The appropriate fractions, as judged by HPLC,were combined and concentrated to provide 77 mg (88%) of product as aclear, colourless oil. HPLC (C8, gradient: 5-80% ACN/H₂O (1-7 min), 80%ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 0.1% TFA) Rt(min) 9.14. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.63-1.06 (m, 211H),1.06-2.11 (m, 789H), 2.32-2.62 (m, 61H), 2.88-3.28 (m, 148H), 3.36 (s,95H), 3.37-4.00 (m, 2920H), 4.17-4.69 (m, 132H), 7.23 (br s, 140H). ¹HNMR indicates ca. 30 Boc-GLFG/dendrimer. Molecular weight isapproximately 53.8 kDa.

(b) Preparation of BHALys[Lys]₃₂α-[αGFLG-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂

To a magnetically stirred mixture ofBHALys[Lys]₃₂[α-GFLG-Boc]₃₂[ε-PEG₁₁₀₀]₃₂ (77 mg, 1.43 μmol) in CH₂Cl₂ (1mL) at room temperature was added a solution of TFA in CH₂Cl₂ (1:1, 2mL). After 3 hours at room temperature the volatiles were removed. Theresulting residue was dissolved in MQ water (15 mL) and concentrated.This procedure was repeated once more. The residue was then dissolved inMQ water (15 mL) and lyophilised to provide 76 mg (99%) of desiredmaterial as a yellowish resin. HPLC (C8, gradient: 5-80% ACN/H₂O (1-7min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min),0.1% TFA) Rt (min)=8.08. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.75-1.04 (m,197H), 1.10-2.09 (m, 480H), 2.45 (m, 56H), 2.88-3.29 (m, 146), 3.35 (s,90H), 3.37-4.05 (m, 2920H), 4.17-4.69 (m, 133H), 7.66 (s, 159H).Theoretical molecular weight of conjugate: 68.9 kDa. ¹H NMR indicatesca. 30 GFLG-NH₂.TFA/dendrimer. Molecular weight is approximately 54.1kDa.

(c) Preparation of BHALys[Lys]₃₂[α-GFLG-Glu-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-GFLG-NH₂.TFA]₃₂[ε-PEG₁₁₀₀]₃₂ (61 mg, 1.13 μmol) andGlu-DTX (42 mg, 45.60 μmol). Purification by SEC (Sephadex, LH-20, MeOH)provided 68 mg (85%) of product as a white solid. HPLC (C8, gradient:5-80% ACN/H₂O (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5%(ACN 13-15 min), 0.1% TFA) Rt (min)=10.16. ¹H NMR (300 MHz, CD₃OD) δ(ppm): 0.85 (s, 173H); 0.99-2.13 (m, 1153H), 2.15-2.62 (m, 312H),2.91-3.27 (m, 128H), 3.35 (s, 93), 3.36-4.00 (m, 2970H), 4.05-4.68 (m,237H), 4.94-5.07 (m, 32H), 5.15-5.47 (m, 76H), 5.52-5.76 (m, 24H),5.97-6.26 (s, 21H), 6.99-7.77 (m, 380H), 7.98-8.24 (m, 48H). Theoreticalmolecular weight of conjugate: 80.4 kDa. ¹H NMR indicates 30 GLFG and 22DTX/dendrimer. Actual molecular weight is approximately 70.6 kDa (25%DTX by weight).

Example 16

Preparation of BHALys[Lys]₃₂[α-GILGVP-Glu-DTX]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[ε-GILGVP-NH.TFA]₃₂[α-PEG₁₁₀₀]₃₂ (52 mg, 0.86 μmol) andGlu-DTX (34 mg, 36 μmol). Purification by SEC (sephadex, LH20, MeOH)provided 59 mg (80%) of desired material as a hygroscopic colourlesssolid. HPLC (C8, gradient: 5-80% ACN/H₂O (1-7 min), 80% ACN (7-12 min),80-5% ACN (12-13 min), 5% ACN (13-15 min), 0.1% TFA buffer) Rt(min)=10.45. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.84-1.91 (m, 1808H), 2.41(s, 287H), 3.12-3.20 (m, 106H), 3.35 (bd, 166H), 3.37-3.90 (m, 2800H),4.10-4.40 (bm, 194H), 4.53 (s, 88H), 4.98-5.03 (m, 35H), 5.24-5.40 (m,80H), 5.60-5.68 (m, 26H), 6.08-6.16 (m, 21H), 7.25-7.88 (m, 288H),8.08-8.16 (m, 86H). Theoretical molecular weight of conjugate: 85.6 kDa.¹H NMR indicates 30 DTX/dendrimer. Actual molecular weight isapproximately 83.2 kDa (29% DTX by weight).

Example 17

Preparation of BHALys[Lys]₃₂[α-GILGVP-Glu-DTX]₃₂[ε-t-PEG₂₃₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-GILGVP-NH₂.TFA]₃₂[ε-t-PEG₂₃₀₀]₃₂ (59 mg, 0.57 μmol) andGlu-DTX (23 mg, 25 μmol) and PyBOP (13 mg, 25 μmol) Purification by SEC(sephadex, LH20, MeOH) provided 65 mg (89%) of desired material as ahygroscopic colourless solid. HPLC (C8, gradient: 5-80% ACN/H₂O (1-7min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min),0.1% TFA buffer) Rt (min)=9.22. ¹H NMR (300 MHz, CD₃OD) δ (ppm):0.86-2.50 (m, 2622H), 3.12-3.20 (m, 80H), 3.35-3.88 (m, 5540H),4.18-4.30 (bm, 263H), 4.50-4.58 (m, 149H), 4.96-5.04 (m, 42H), 5.24-5.38(m, 77H), 5.62-5.68 (m, 29H), 6.08-6.14 (m, 28H), 7.25-7.70 (m, 234H),8.10-8.15 (m, 63H). Theoretical molecular weight of conjugate: 127.3kDa. ¹H NMR indicates 27 DTX/dendrimer. Actual molecular weight isapproximately 123.7 kDa (18% DTX by weight).

Example 18

Preparation of BHALys[Lys]₃₂[α-PEG₁₁₀₀]₃₂[ε-TDA-DTX]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[—NH₂.TFA]₃₂[α-PEG₁₁₀₀]₃₂ (57.5 mg, 1.34 μmol) and TDA-DTX(52.3 mg, 56 μmol). Purification by SEC (sephadex, LH20, MeOH) provided70 mg (92%) of desired material as a hygroscopic colourless solid. HPLC(C8, gradient 5-80% ACN/H₂O (1-7 min), 80% ACN (7-12 min), 80-5% ACN(12-13 min), 5% ACN (13-15 min), 0.1% TFA buffer) Rt (min)=9.89. ¹H NMR(300 MHz, CD₃OD) δ (ppm): 1.06-1.95 (m, 784H), 2.36-2.55 (m, 168H),3.04-3.23 (m, 48H), 3.33 (s, 84H), 3.35-3.89 (m, 2800H), 4.13-4.40 (m,118H), 5.23-5.40 (m, 72H), 5.59-5.66 (m, 24H), 6.06-6.16 (m, 23H),7.25-7.65 (m, 234H), 8.10-8.12 (m, 52H). Theoretical molecular weight ofconjugate: 68.9 kDa. ¹H NMR indicates 27 DTX/dendrimer. Actual molecularweight is approximately 64.4 kDa (34% DTX by weight).

Example 19

Preparation of BHALys[LYS]₃₂[α-TDA-DTX]₃₂[ε-PolyPEG₂₀₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₂₀₀₀]₃₂ (88.6 mg, 1.2 μmol) and TDA-DTX(49.3 mg, 52 μmol). Purification by SEC (sephadex, LH20, MeOH) provided95 mg (80%) of desired material as a hygroscopic colourless solid. HPLC(C8, gradient: 45-85% ACN/H₂O (1-7 min), 85% ACN (7-12 min), 85-45% ACN(12-13 min), 45% ACN (13-15 min), 0.1% TFA buffer) Rf (min)=6.29 min. ¹HNMR (300 MHz, CD₃OD) δ (ppm): 0.82-1.96 (m, 2076H), 2.36-2.54 (m, 314H),3.10-3.24 (m, 125H), 3.35-3.89 (m, 6300H), 4.96-5.04 (m, 35H), 525-5.45(m, 79H), 5.60-5.70 (m, 29H), 6.06-6.18 (m, 24H), 7.20-7.75 (m, 269H),8.06-8.16 (m, 52H). Theoretical molecular weight of conjugate: 101.1kDa. ¹H NMR indicates 27 DTX/dendrimer. Actual molecular weight isapproximately 95.5 kDa (23% DTX by weight). Particle sizing usingDynamic Light Scattering shows a range of concentration dependentaverages of 10.9-15.5 nm.

Example 20

Preparation of BHALys[Lys]₃₂[α-DGA-testosterone]₃₂[ε-PEG₁₁₀₀]₃₂

(a) Preparation of DGA-Testosterone

Prepared using Procedure B above, using testosterone (256 mg, 0.88mmol), pyridine (10 mL) as the solvent and diglycolic anhydride (1.02 g,8.8 mmol) as the linker. Purification by preparatory HPLC (BEH 300Waters XBridge C18, 5 μM, 30×150 mm, 40-90% ACN/water, no buffer, RT=62min) to give the desired compound 241 mg (67% yield) as an off whitehygroscopic solid. LCMS (C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN(7-9 min), 90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% TFA)Rt(min)=5.61. ESI (−ve) observed [M−H]⁻=403.29. Calculated forC23H₃₁O₆=403.21 Da. ¹H NMR (300 MHz, CD₃OD) δ (ppm) 0.88 (s, 3H, CH₃),0.93-1.23 (m, 3H), 1.24 (s, 3H, CH₃), 1.25-2.58 (br m, 16H), 4.18 (s,2H, CH₂), 4.23 (s, 2H, CH₂), 4.70 (m, 1H, CH), 5.71 (s, 1H, CH).

(b) Preparation of BHALys[Lys]₃₂[α-DGA-Testosterone]₃₂[α-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂(α-NH₂.TFA)₃₂(ε-PEG₁₁₀₀)₃₂ (30 mg, 0.75 μmol) andDGA-Testosterone (19 mg, 47 μmol). Purification by SEC (LH20, eluent:methanol) provided 15 mg (39%) as an off-white solid. HPLC (C8,gradient: 30-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-30% ACN (9-11min), 30% ACN (11-15 min), 10 mM ammonium formate) Rt(min)=9.41. ¹H NMR(300 MHz, CD₃OD) δ (ppm) 0.79 (s, 80H, CH₃), 0.81-2.42 (br m, 1101H),3.08 (m, 116H, CH₂), 3.26 (s, 98H, CH₂), 3.37-3.81 (m, 2800H, CH₂),3.95-4.47 (m, 173H, CH), 4.61 (m, 29H, CH), 5.62 (s, 29H, CH), 6.08 (m,1H, CH), 7.17 (m, 10H, ArH). Theoretical molecular weight of conjugate:52.4 kDa. ¹H NMR indicates 29 testosterone/dendrimer. Actual molecularweight is approximately 51.2 kDa (16% testosterone by weight).

Example 21

Preparation of BHALys[Lys]₃₂[α-DGA-Testosterone]₃₂[ε-PEG₅₇₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂(α-NH₂.TFA)₃₂(ε-PEG₅₇₀)₃₂ (40 mg, 1.33 μmol) in DMF (2 mL)and DOA-Testosterone (43 mg, 106 μmol). Purification by SEC (LH20,eluent: methanol) provided 22.1 mg (40% yield) as a white hygroscopicsolid. HPLC (C8, gradient: 30-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min),80-30% ACN (9-11 min), 30% ACN (11-15 min), 10 mM ammonium formate) Rt(min)=9.99. ¹H NMR (300 MHz, CD₃OD) δ (ppm) 0.89 (s, 96H, CH₃),0.90-2.63 (br m, 1214H), 3.36 (m, 125H, CH₂), 3.36 (s, 100H, CH₃),3.45-3.97 (m, 1472H, CH₂), 4.05-4.62 (m, 218H), 4.71 (m, 37H, CH), 5.72(s, 31H, CH), 6.18 (m, 1H, CH), 7.17 (m, 10H, ArH). Theoreticalmolecular weight of conjugate: 42.5 kDa. ¹H NMR indicates 31testosterone/dendrimer. Actual molecular weight is approximately 42.1kDa (21% testosterone by weight).

Example 22

Preparation of BHALys[Lys]₃₂[α-Glu-testosterone]₃₂[ε-PEG₁₁₀₀]₃₂

(a) Preparation of Glu-Testosterone

Prepared using Procedure B above, using testosterone (100 mg, 0.35mmol), pyridine (6 mL) as the solvent and glutaric anhydride (396 mg,3.5 mmol) as the linker. Purification by preparatory HPLC (BEH 300Waters XBridge C18, 5 μM, 30×150 mm, 40-90% ACN/water, no buffer, RT 62min) to give the desired compound 86 mg (86%) as an off whitehygroscopic solid. LCMS (C8, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN(7-9 min), 90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% TFA) Rt(min)=6.40. ESI (+ve) observed [M+H]⁺=403.29. Calculated forC₂₄H₃₅O₅-403.25 Da. ¹H NMR (300 MHz, CD₃OD) δ (ppm) 0.89 (s, 3H, CH₃),0.93-1.23 (m, 3H), 1.24 (s, 3H, CH₃), 1.36-2.57 (br m, 22H), 4.62 (m,1H, CH), 5.71 (s, 1H, CH).

(b) Preparation of BHALys[Lys]₃₂[α-Glu-Testosterone]₃₂[ε-PEG₁₁₀₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂(α-NH₂.TFA)₃₂(ε-PEG₁₁₀₀)₃₂ (30 mg, 0.75 μmol) in DMF (2 mL)and Glu-Testosterone (19 mg, 47 μmol). Purification by SEC (LH20,eluent: methanol) provided 18.1 mg (47%) of the desired product as anoff-white solid. HPLC (C8, gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN(7-9 min), 80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mM ammoniumformate) Rt (min)=7.22. ¹H NMR (300 MHz, CD₃OD) δ (ppm) 0.88 (s, 87H,CH₃), 0.89-2.61 (br m, 1225H), 3.17 (m, 110H, CH₂), 3.36 (s, 101H, CH₃),3.46-3.98 (m, 2800H, CH₂), 4.34 (m, 59H, CH), 4.61 (m, 30H, CH), 5.72(s, 29H, CH), 6.18 (m, 1H, CH), 7.28 (m, 12H, ArH). Theoreticalmolecular weight of conjugate: 52.3 kDa. ¹H NMR indicates 29testosterone/dendrimer. Actual molecular weight is approximately 51.1kDa (16% testosterone by weight).

Example 23

Preparation of BHALys[Lys]₃₂[α-Glu-Testosterone]₃₂[ε-PEG₅₇₀]₃₂

Prepared using Procedure C above, usingBHALys[Lys]₃₂(α-NH₂.TFA)₃₂(ε-PEG₅₇₀)₃₂ (30 mg, 1 μmol) in DMF (2 mL) andExample 22(a), Glu-Testosterone (26 mg, 64 μmol). Purification by SEC(LH20, eluent: methanol) provided 19.8 mg (47% yield) of the desiredproduct as a white solid product. HPLC (C8, gradient: 40-80% ACN/H₂O(1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40% ACN (11-15min), 10 mM ammonium formate) Rt (min)=8.93. ¹H NMR (300 MHz, CD₃OD) δ(ppm): 0.88 (s, 96H, CH₃), 0.89-2.59 (br m, 1423H), 3.16 (m, 127H, CH₂),3.26 (m, 135H, CH₃), 3.65-3.92 (m, 1472H, CH₂), 4.24 (m, 66H, CH), 4.52(m, 39H, CH), 5.62 (s, 32H, CH), 6.09 (m, 1H, CH), 7.19 (m, 10H, ArH).Theoretical molecular weight of conjugate: 42.5 kDa. ¹H NMR indicates 32testosterone/dendrimer. Actual molecular weight is approximately 42.5kDa (21% testosterone by weight).

Example 24

Preparation of BHALys[Lys]₃₂[α-Glu-SB]₃₂[ε-PEG₁₁₀₀]₃₂ SB=Salbutamol

(a) Preparation of Glu-SB

Prepared using Procedure B above, using SB (100 mg, 0.42 mmol) andglutaric anhydride (62 mg, 0.54 mmol) as the linker. Preparative HPLC(BEH 300 Waters XBridge C18, 5 μM, 30×150 mm, gradient: 5% ACN/H₂O (1-5min), 5-60% ACN (5-40 min), 60% ACN (40-45 min), 60-5% ACN (45-50 min),5% ACN (50-60 min), 0.1% TFA, Rt=27 min) provided 50 mg (34%) of thedesired product as a white solid. HPLC (C18, gradient: 5-60% ACN/H₂O(1-10 min), 60% ACN (10-11 min), 60-5% ACN (11-13 min), 5% ACN (13-15min), 10 mM ammonium formate) Rt (min)=6.67. ESI (+ve) observed[M+H]⁺=354. Calculated for C₁₈H₂₇NO₆=353.18 Da. ¹H NMR (300 MHz, CD₃OD)δ (ppm): 1.41 (s, 9H), 1.92 (t, J=7.2 Hz, 2H), 2.37 (t, J=7.5 Hz, 2H),2.45 (t, J=7.2 Hz, 2H), 3.01-3.18 (m, 2H), 5.18 (s, 2H), 6.87 (d, J=8.4Hz, 1H), 7.27 (dd, J=8.4 and 2.1 Hz, 1H), 736 (d, J=2.4 Hz, 1H).

(b) Preparation of BHALys[Lys]₃₂[α-Glu-SB]₃₂[ε-PEG₅₇₀₀]₃₂

Prepared using Procedure C above, usingBHA[Lys]₃₂[α-NH₂.TFA]₃₂[ε-PEG₅₇₀]₃₂ (26 mg, 0.86 μmol) and Glu-SB (17mg, 48.2 μmol). Purification by SEC (sephadex, LH20, MeOH) provided 25mg (76%) of desired material as a white solid. HPLC (C8, gradient:40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min), 80-40% ACN (9-11 min), 40%ACN (11-15 min), 10 mM ammonium formate) Rt (min)=5.81. ¹H NMR (300 MHz,CD₃OD) δ (ppm): 1.03-2.02 (m, 738H), 2.25-2.58 (m, 180H), 2.97-3.29 (m,167H), 3.40-3.94 (m, 1469H), 4.12-4.50 (m, 74H), 5.04 (s, 55H), 6.90 (d,J=8.1 Hz, 27H), 7.28 (d, J=8.1 Hz, 27H), 7.36 (m, 27H). Theoreticalmolecular weight of conjugate: 37.8 kDa. ¹H NMR indicates 27salbutamol/dendrimer. Actual molecular weight is approximately 36.1 kDa(18% salbutamol by weight).

Targeted Constructs

Example 25

Preparation of 4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂

(a) Preparation of4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-NHBOC)(ε-PEG₁₁₀₀)]₃₂

To a magnetically stirred solution of L-lysine-(α-NHBOC)(ε-PEG₁₁₀₀) (614mg, 456 μmol) in anhydrous DMF (2.5 mL) was added PyBOP (246 mg, 473μmol) followed by a solution of4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[NH₂.TFA]₃₂ (91 mg, 10.6μmol) and DIPEA (235 μL, 1.35 mmol) in anhydrous DMF (2.5 mL). After 16hours at room temperature the reaction was concentrated in vacuo and theresidue purified by ultrafiltration (PALL Minimate Cartridge 10 kDamembrane) to provide the target compound as an off-white sticky solid,433 mg (86%). LCMS (C8 Waters X-Bridge, gradient: 40-90% ACN/H₂O (1-7min), 90% ACN (7-9 min), 90-40% ACN (9-11 min), 40% ACN (11-15 min),0.1% Formic Acid) Rt (min)=5.17.

(b) Preparation of4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-NH₂.TFA)(ε-PEG₁₁₀₀)]₃₂

A solution of4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-NHBOC)(ε-PEG₁₁₀₀)]₃₂(431 mg, 9.10 μmol) in TFA/DCM (5 mL/7 mL) was left stirring for 4 h.After this time the reaction mixture concentrated and the resultingresidue azeotroped with water (2×10 mL) to provide the target compoundas a pale yellow oil, 435 mg (100%). LCMS (C18 Waters X-Bridge,gradient: 5-60% ACN/H₂O (1-10 min), 60% ACN/H₂O (10-14 min), 60-5%ACN/H₂O (14-16 min), 0.1% TFA) Rt=10.65. ¹H NMR (300 MHz, D₂O) δ (ppm):1.21-2.04 (m, 376H), 2.51-2.56 (m, 71H), 3.12-3.30 (m, 115H), 3.40 (s,96H), 3.45-3.90 (m, 3077H), 3.91-4.42 (m, 62H), 7.25 (d, J 8.7 Hz, 2H),7.88 (d, J 8.7 Hz, 2H).

(c) Preparation of4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂

The construct was prepared using Procedure C above, using4-azidobenzamide-PEG₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-NH₂.TFA)(δ-PEG₁₁₀₀)]₃₂(104 mg, 2.18 μmol) and DTX-PSSP (94 mg, 94.0 μmol). Purification by SECprovided 133 mg (97%) of the desired material as a pale yellow, viscousoil. LCMS (C18 Waters X-Bridge, gradient: 5-60% ACN/H₂O (1-10 min), 60%ACN/H₂O (10-11 min), 60-5% ACN/H₂O (11-13 min), 0.1% Formic acid) Rt(min)=7.59. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.88-2.05 (m, 1080H),2.16-2.56 (m, 212H), 2.60-3.26 (m, 363H), 3.35-3.41 (m, 129H), 3.50-3.94(m, 3110H), 4.00-4.60 (134H), 4.93-5.10 (m, 28H), 5.20-5.46 (m, 73H),5.54-5.80 (m, 24H), 5.95-6.30 (m, 23H), 7.14-7.91 (m, 268H). Theoreticalmolecular weight of conjugate: 75.7 kDa. ¹H NMR indicates 26DTX/dendrimer, therefore actual molecular weight is approximately 69.8kDa (37% DTX by weight).

Example 26

Preparation ofbiotin-triazolobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂

The construct was prepared using Procedure D above, using4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂(42.5 mg, 674 nmol) and biotin-alkyne (0.4 mg, 1.35 μmol). Purificationby SEC provided the target compound as an off-white solid, 39 mg (91%).LCMS (C18 Waters X-Bridge, gradient: 5-60% ACN/H₂O (1-10 min), 60′/eACN/H₂O (10-11 min), 60-5% ACN/H₂O (11-13 min), 0.1% Formic acid) Rt(min)=7.04. ¹H NMR (300 MHz, CD₃OD) δ (ppm): 0.92-2.02 (m, 982H),2.10-3.25 (m, 1027H), 3.35-3.42 (m, 128H), 3.49-3.98 (m, 3180H),4.07-4.69 (m, 131H), 4.96-5.11 (m, 27H), 5.15-5.50 (m, 72H), 5.55-5.80(m, 24H), 5.98-6.23 (m, 23H), 7.14-8.25 (m, 277H), 8.54-8.56 (m, 1H).

Example 27

Preparation ofLyP-1-triazolobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂

LyP-1 (Supplied by AusPep Pty Ltd).

The construct was prepared using Procedure D above, using4-azidobenzamide-PEO₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂(44.2 mg, 701 nmol) LyP-alkyne (185 μL of a 10 mg/mL solution in H₂O,1.05 μmol). Purification by SEC provided a bright pink, sticky solid, 46mg (102%), as a ca. mixture of 60:40LyP-triazolobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂/4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]N₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂.LCMS (C8 Waters X-Bridge, gradient: 40-90% ACN/H₂O (1-7 min), 90% ACN(7-9 min), 90-40% ACN (9-11 min), 40% ACN (11-15 min), 0.1% Formic Acid)Rt (min)=6.07 (LyP-Dendrimer conjugate); 7.10 (Azido-Dendrimer startingmaterial).

Example 28

Preparation ofdeslorelin-triazolobenzamide-PEG₁₂NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂

The construct was prepared using Procedure D above, using4-azidobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂(41.7 mg, 662 nmol) and deslorelin-alkyne (130 μL of a 10 mg/mL solutionin H₂O, 993 nmol). Purification by SEC provided a pale yellow, stickysolid, 43 mg (100%), as a ca. mixture of 70:30deslorelin-triazolobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂/4-azidobenzamide-PEO₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂. LCMS (C8 Waters X-Bridge, gradient: 40-90% ACN/H₂O (1-7min), 90% ACN (7-9 min), 90-40% ACN (9-11 min), 40% ACN (11-15 min),0.1% Formic Acid) Rt (min)=6.42 (Deslorelin-Dendrimer conjugate); 7.11(Azido-Dendrimer starting material).

Example 29

Preparation Antibody-Dendrimer Conjugation Using Streptavidin as aJoining Unit

To a solution of Alexa Fluor® 750 Streptavidin (Av) (0.1 μg/mL) inphosphate-buffered saline (PBS, 2 mL) was added Abcam #ab24293 Anti-EGFRantibody biotin (Ab) (30 μL of 10 μg/mL stock solution). To thisreaction solution was added a solution ofbiotin-triazolobenzamide-PEG₁₂-NEOEOEN[SuN(PN)₂][Lys]₁₆[Lys(α-PSSP-DTX)(ε-PEG₁₁₀₀)]₃₂(DTX-D) in PBS (5 μL of 1.0 μg/mL stock solution). The mixture was leftstirring for 10 s and the above procedure of adding Ab and DTX-D to theAv solution was repeated in total of 8 times. Finally the reaction wasquenched using 50 μg/mL of Biotin, (Sigma Aldrich, #B4501-1G), and afterincubating for 5 min, 1 mL of the sample was precipitated with 50 μL ofProtein G agarose. Confirmation of successful conjugation wasdemonstrated using SDS-PAGE with a new band assigned to the conjugateappearing at 260 kDa and HPLC (column: X Bridge C8, 3.5 μm 3.0×100 mm,detection wavelength=243 nm, 10 μL injections and run gradient: 5-80%ACN/H₂O, 0.1% TFA for 15 min Rt (min)=1.40 biotin, 5.83 (Target Ab-DTX-Dconjugate); 7.24 (unreacted Ab), 9.84 (unreacted DTX-D).

Example 30

Preparation of an Antibody Activated with an Azide Joining Unit

A solution of coupling buffer (0.1 M sodium acetate+0.15 M NaCl, pH 5.5)was prepared and used to make up stock solutions for the followingreaction. Solid sodium meta-periodate (2.1 mg) was dissolved in couplingbuffer (0.5 mL) and then was added to a solution of Her2 mAb* (25 μg)also diluted in coupling buffer (0.5 mL). The reaction mixture wasincubated at room temperature (RT) in the dark for 45 min. Unreactedmaterial was removed by centrifugal filter units (MW cut off 50 kDa). Toa portion of the oxidised mAb solution (0.3 mL) was added a stocksolution of a azide containing joining unit (JU)(NH₂—O—C₄H₈—NH-(PEG)₁₂-N₃ ¥, 0.2 mL; 1 mg/mL in PBS), followed byaniline (5 μL). The reaction was mixed and left for 24 h at RT. Afterthis time the mAb-JU conjugate was separated from unreacted material bycentrifugal filter units.

^(¥)In a similar manner other joining units could also be installed ontothe antibody, e.g. NH₂—O—C₄H₈—NH-(PEG)₁₂-benzylazide,NH₂—O—C₄H₈—NH-(PEG)₁₂-DBCO and NH₂—O—C₄H₈—NH-(PEG)₁₂-maleimide.

*In this example Her2 mAb is utilised however, in a similar fashionother antibodies could also be utilised. In addition to utilising otheractivating chemistry's e.g. partial reduction of dithiane groups withinthe antibody followed by capture with maleimide containing joining units

Example 31

Conjugation of the Activated Antibody with a Drug Loaded Dendrimer

To a solution of the azide activated mAb-JU from Example 30 above couldbe added a solution of a drug loaded dendrimer suitably functionalisedwith a reactive alkyne, such as DBCO. The reaction could be monitoredfor completion using HPLC and the desired product could be isolated byeither SEC chromatography or prep HPLC using standard protocols.

^(¥)In a similar manner other dendrimer activating units could also beinstalled onto the unique point of attachment in the dendrimer, e.g.azide and maleimide.

Example 32

Water Solubility Study on Drug Loaded Dendrimers:

Protocol:

To 30 mg of dendrimer (freeze-dried from water) was added 100 μL ofdeionised water. After mixing for 10 minutes, additional aliquots ofwater (10-30 μL per addition) were added with vortexing and incubationfor 10 mins until full dissolution was obtained. This amount isrepresented in Table 1 as the water solubility of the dendrimer. Theequivalent drug solubility is determined by multiplying the % drugloading/100 and is represented in Table 1 (column 3) as Equivalent drugsolubility on dendrimer. Finally, the fold increase is obtained bydividing the Equivalent drug solubility on dendrimer by the solubilityof the drug and is represented in Table 1 (column 4).

TABLE 1 3 2 Equivalent 4 Water drug Fold solubility solubility increase1 of dendrimer on dendrimer in drug Example (mg/mL) (mg/mL) solubility 1(b)* 186 24 4800 2 (b)* 57 14 2800 3 (b)* 89 23 5600 4 (c)* 109 22 44005 (b)* 214 75 4000 6 (b)* 100 32 6400 7 (b)* 91 25 5000 8 (c)* 131 418200 9 (b)* 63 20 4000 10 (b)* 138 43 8600 12 (b)* 15 3 10000 14 (c)*183 57 11400 15 (c)* 180 45 9000 16* 205 59 11800 17* 373 67 13400 19*477 109 21900 20 (b)¥ >75 11.5 482 21¥ >81 14.8 618 22 (b)¥ >89 14.7 61023¥ >125 26.6 1109 *drug = docetaxel. The solubility of docetaxel and inwater is 5 μg/mL ¥drug = testosterone: The solubility of testosterone inwater is 24 μg/mL.

Example 33

Plasma Stability Study on Dendrimers:

Protocol:

To 0.5 mL of mouse plasma was added 0.1 mL of dendrimer solution (2mg/mL, drug equivalent in saline). The mixtures were vortexed (30 s)then incubated at 37° C. At various timepoints (0.5, 2.5, 4.5, 22 hours)0.1 mL aliquots were removed and added to 0.2 mL ACN. The resultingmixtures were vortexed (30 s), centrifuged (10 min, 4° C.) filtered andanalysed by HPLC (C8, 3.9×150 mm, 5 μm, wavelength=243 nm, 10 μLinjections, gradient: 40-80% ACN/H₂O (1-7 min), 80% ACN (7-9 min),80-40% ACN (9-11 min), 40% ACN (11-15 min), 10 mM ammonium formate, pH7.40) which when compared against a standard (2 mg//mL) provided theconcentration of free docetaxel in the sample.

TABLE 2 Docetaxel release in plasma. Results are shown as a percentageof total docetaxel. Example Time Compound 0.5 2.5 4.5 22 Exp 3(b) 8.532.5 52.5 73 Exp 10(b) 10 21 28.5 75 Exp 7(b) 20.5 32 32.5 71.5 Exp14(c) 4 9 16 70 Exp 8(c) 4.5 13.5 17.5 43 Exp 6(b) 7.5 9 13 23.5 Exp4(c) 1.5 10 18.5 17.5 Exp 2(b) 5 8 11.5 15.5 Exp 1(b) 0 3 7.5 14.5 Exp15(c) 0 5 8 45 Exp 5(b) 0 0 0 4 Exp 9(b) 0.5 1.5 1 1 Exp 16 0 0 0 0 Exp17 0 0 0 1

Example 34

Cell Growth Inhibition Studies SRB Assay

Cell growth inhibition was determined using the Sulforhodamine B (SRB)assay [Voigt W. “Sulforhodamine B assay and chemosensitivity” MethodsMol. Med. 2005, 110, 39-48.] against various cancer cell lines after 72hours with each experiment run in duplicate. GI₅₀ is the concentrationrequired to inhibit total cell growth by 50%, as per NCI standardprotocols.

All solutions were prepared in saline (except docetaxel which was madein ethanol). All solutions were stored at −20° C. All values were basedon the equivalent drug loading. The results shown in Table 3 are theaverage of experiments run in duplicate in nanomolar range.

TABLE 3 Growth Inhibition Studies. GI₅₀ Values (nM) Exp Exp Exp Exp ExpExp Exp Exp Exp Exp Exp Cell line Docetaxel 1 (b) 3 (b) 4 (c) 5 (b) 13(b) 2 (b) 6 (b) 7 (b) 8 (c) 9 (b) 10 (b) PC-3 2.5 17 4.5 21.5 160 288109.5 10.5 6.5 9.5 617.5 9.5 (Prostate) DU145 2.5 11.5 4 12 148 99(Prostate) HCT116 0.7 8.5 1 9 85.5 30.5 (Colon) ES2 5 16.5 4 8.6 115.548 115.5 12.5 8 12 888 10.5 (Ovarian) HT29 1.5 12.5 2 9.5 97.5 117(Colon) H460 1.5 13 8 11 106 127 73 11 4.5 7 365 6.5 (Lung) A549 3.5 133.5 8.5 56.5 73 (Lung) MDA-MB- 3.5 11.5 0.5 6.5 50.5 50.5 231 (Breast)A2058 2 9.5 2 8 71.5 100.5 (Melanoma) MCAS 7 29 7 20 252.5 117 (Ovarian)

Example 35

Half Maximal Inhibitory Concentration (IC₅₀) Using the MTT Assay

The IC₅₀ using the MTT assay [Wilson, Anne P. (2000). “Chapter 7:Cytotoxicity and viability”. In Masters, John R. W. Animal Cell Culture:A Practical Approach. Vol. 1 (3rd ed.). Oxford: Oxford University Press]was determined against various cancer cell lines after 72 hours. Theresults are shown in Table 4.

TABLE 4 Half Maximal Inhibitory Concentration Studies (IC₅₀). IC₅₀Values(nM) Cell line Exp 14 (c) Exp 15 (c) Exp 17 Exp 18 Exp 19 A549 1.5 8.1159.7 20.3 7.7 H460 4.3 31.8 603.3 7.5 23.7 HCT-116 2.6 7.2 215.7 2.96.5 HT-29 0.5 5.7 85 1.8 5.9 A2780 4.6 13.6 291 5.7 6.3 MCF-7 0.5 8.393.7 3.3 6.3 DU-145 7.3 29.5 290 11.6 15.5 PC-3 3.8 11.8 358.7 5.9 7.4

Example 36

Maximum Tolerated Dose (MTD) Study

Groups of female Balb/c mice were administered an intravenous injectionof dendrimer (0.1 ml/10 g body weight) or docetaxel (0.05 ml/10 g bodyweight) once weekly for 3 weeks (day 1, 8 and 15). Mice were weigheddaily and watched for signs of toxicity. Animals were monitored for upto 10 days following the final drug dose. Any mice exceeding ethicalendpoints (≧20% body weight loss, poor general health) were immediatelysacrificed and observations were noted. The results shown in Table 5demonstrate that drug conjugated to the dendrimer increases thetolerated dose. More than twice the dose of docetaxel could be safelyadministered using drug dendrimer construct compared to docetaxel alone.

TABLE 5 Drug doses tested and maximum tolerated dose identified Dosestested (mg/kg Tolerated dose (mg/kg Drug docetaxel equivalents)docetaxel equivalents) Docetaxel 15, 20, 25, 30 15 Example 3 (b) 15, 20,23, 25, 30 20 Example 8 (c) 15, 20, 25, 30, 32, 35 32 Example 4 (c) 20,25, 30 20

Example 37

Xenograft MDA-MB-231 Efficacy Study

Female Balb/c nude mice (Age 7 weeks) were inoculated subcutaneously onthe flank with 3.5×106 MDA-MB-231 cells in PBS:Matrigel (1:1). Thirteendays later 50 mice with similar sized tumours (˜110 mm³) were randomisedinto 5 groups. Each treatment group was administered one of thefollowing doses: saline; docetaxel (15 mg/kg); Exp. 3 (b) (20 mg/kg);Exp. 8 (c) (32 mg/kg). All treatments were administered intravenouslyonce weekly for three weeks (day 1, 8 and 15) at 0.1 mL/10 g body weightexcept docetaxel which was given at 0.05 mL/10 g body weight. Theexperiment was ended on day 120 or earlier if an ethical endpoint wasmet. Results shown in Table 6 show that the dendrimer constructs weremore effective in suppressing tumour growth for longer.

TABLE 6 Xenograft efficacy study showing mean tumour volume mm³ overtime Mean tumour Volumne mm³ (sd) Day Vehicle Docetaxel Exp 3 (b) Exp 8(c) 1 112.35 111.94 111.74 111.73 (6.31), (6.41), (6.65), (6.41), n = 10n = 10 n = 10 n = 10 9 426.55 135.57 84.02 108.86 (24.11), (18.85),(6.33), (9.31), n = 10 n = 10 n = 10 n = 10 19 1337.61 49.92 28.26 30.59(18.4), (11.61), (1.91), (1.64), n = 4 n = 10 n = 10 n = 10 29 ** 18.8110.46 11.58 (2.09), (0.5), (1.2), n = 10 n = 8 n = 9 40 10.75 5.92 5.75(1.95), (1.31), (0.92), n = 10 n = 5 n = 8 61 95.94 4 4 (33.08), (0),(0), n = 10 n = 4 n = 8 81 478.67 0.5 0.5 (169.27), (0), (0), n = 7 n =4 n = 8 100 974.83 0.5 1.67 (302.59), (0), (0.74), n = 3 n = 4 n = 6 120** 0.37 16.2 (0.12), (10.24), n = 4 n = 6 ** No data due to ethicalendpoint reached. n = number of animals per dosing group

Example 38

Xenograft MDA-MB-231 Toxicity Study

A total of twenty Female Balb/c nude mice (Age 7 weeks) were preparedwith subcutaneous tumours as outlined above. The 20 mice were randomisedinto 5 groups of four mice (mean tumour volume ˜90 mm³). Animals wereeye bled in the morning for baseline blood cell counts and then drugdosing commenced later that day (day 1). Drug dosing was performed ondays 1, 8 and 15 at the previously determined MTD doses: docetaxel (15mg/kg); Exp. 3 (b)(20 mg/kg); Exp. 8 (c) (32 mg/kg); Exp. 4 (b) (20mg/kg). A second eye bleed was performed on day 11 (Table 7 A-C). Micewere killed one day following the final drug dose (day 16). Histologyweights of tissues at day 16 are shown in Table 8.

TABLE 7 A White Blood Cell analysis at days 1 and 11. Mean WBC (sd) ×10⁹ cells/L PBS docetaxel Exp. 3 (b) Exp. 8 (c) Exp.4 (b) Day 1 5.765.79 5.79 6.59 4.95 (0.31) (1.01) (1.53) (0.62) (2.25) Day 11 8.57 3.993.99 4.27 5.37 (1.94) (0.93) (0.29) (0.35) (1.72)

TABLE 7 B Results of Neutrophil Analysis at days 1 and 11. MeanNeutrophils (sd) × 10⁹ cells/L PBS docetaxel Exp. 3 (b) Exp. 8 (c))Exp.4 (b) Day 1 1.53 0.86 1.01 0.93 1.07 (1.12) (0.26) (0.53) (0.51)(0.57) Day 11 2.84 0.85 1.84 1.76 1.27 (0.62) (0.12) (0.18) (0.15)(0.64)

TABLE 7 C Results of Lymphocyte analysis at days 1 and 11. MeanLymphocytes (sd) × 10⁹ cells/L PBS docetaxel Exp. 3 (b) Exp. 8 (c) Exp.4(b) Day 1 5.76 5.79 5.79 6.59 4.95 (0.31) (1.01) (1.53) (0.62) (2.25)Day 11 8.57 3.99 3.99 4.27 5.37 (1.94) (0.93) (0.29) (0.35) (1.72)

TABLE 8 Organ Weights at Completion of Toxicity Experiment. Exp. 3 Exp.8 Exp. 4 PBS Docetaxel (b) (c) (b) Mean Tumour Weights (g) 0.832 0.0480.020 0.033 0.079 (sd) (0.277) (0.010) (0.008) (0.011) (0.048) MeanSpleen Weights (g) 0.149 0.068 0.077 0.092 0.087 (sd) (0.022) (0.003)(0.011) (0.019) (0.027) Mean Liver Weights (g) 0.838 0.793 0.763 0.7800.762 (sd) (0.058) (0.087) (0.090) (0.103) (0.096)

Example 39

Pharmacokinetic Analysis

The plasma half-lives of tritium labelled docetaxel and the constructfrom Experiment 8 (c) (prepared using tritium labelled docetaxel) afterIV administration into rats were determined (Kaminskas, L. M., Boyd, B.J., Karellas, P., Krippner, G. Y., Lessene, R., Kelly, B and Porter, C.J. H. “The Impact of Molecular Weight and PEG Chain Length on theSystemic Pharmacokinetics of PEGylated Poly-L-Lysine Dendrimers”Molecular Pharm. 2008, 5, 449-463). Results showed docetaxel was clearedfrom plasma with a half-life of <1 hour as expected whilst Exp 8 (c)construct displayed reduced plasma clearance with a half-life ofapproximately 30 hour.

What is claimed is:
 1. A macromolecule comprising: i) a dendrimercomprising a core and at least one generation of lysine residue buildingunits, the outermost generation of building units having surface aminogroups; ii) a first terminal group covalently attached to a firstsurface amino group of a building unit, comprising a residue ofdocetaxel (DTX); iii) a second terminal group covalently attached to asecond surface amino group of a building unit, comprising apharmacokinetic modifying agent, wherein the pharmacokinetic modifyingagent is a polyethylene glycol (PEG); wherein the first terminal groupis covalently attached to the surface amino group of the dendrimerthrough a diacid linker, the diacid linker comprising a2,2′-thiodiacetic acid residue; wherein the diacid linker forms an esterbond with a hydroxyl group of the DTX and an amide bond with the surfaceamino group; or a pharmaceutically acceptable salt thereof.
 2. Themacromolecule of claim 1, wherein the polyethylene glycol has amolecular weight in the range of 1000 to 2500 Da.
 3. The macromoleculeof claim 1, wherein the polyethylene glycol has an average molecularweight of 2200 Da.
 4. The macromolecule of claim 1, wherein the firstterminal group is covalently attached to an α-amino surface group of alysine residue building unit, and the second terminal group iscovalently attached to an ε-amino surface group of a lysine residuebuilding unit.
 5. The macromolecule of claim 1, wherein the firstterminal group is covalently attached to an ε-amino surface group of alysine residue building unit, and the second terminal group iscovalently attached to an α-amino surface group of a lysine residuebuilding unit.
 6. The macromolecule of claim 1, wherein the dendrimerhas 3 to 6 generations of building units.
 7. The macromolecule of claim1, wherein the dendrimer has 5 generations of building units.
 8. Themacromolecule of claim 1, wherein the first terminal group and thesecond terminal group are present in about a 1:1 ratio.
 9. Themacromolecule of claim 1, wherein the first terminal group is attachedto greater than 44% of the surface amino groups.
 10. The macromoleculeof claim 1, wherein the second terminal group is attached to greaterthan 44% of the surface amino groups.
 11. The macromolecule of claim 1,wherein the macromolecule comprises a third terminal group which is ablocking group, or a targeting group.
 12. The macromolecule of claim 1,wherein the macromolecule has a particulate size of less than 1000 nmand/or has a molecular weight of at least 30 kDa.
 13. The macromoleculeof claim 1, wherein the macromolecule releases greater than 50% of theDTX in between 2 hours and 48 hours, as measured in vitro in plasma at37° C.
 14. A pharmaceutical composition comprising the macromolecule ofclaim 1 and a pharmaceutically acceptable carrier.
 15. Thepharmaceutical composition of claim 14, wherein one or more of thefollowing apply: (a) the composition is substantially free ofpolyethoxylated castor oil and polysorbate 80; (b) the plasma half-lifeof released DTX is at least two times the half-life of native DTX(taxotere).
 16. A method of treating or suppressing the growth of acancer in a subject comprising administering an effective amount of amacromolecule according to claim 1, wherein the dose is equivalent to 1mg to 100 mg per kg of DTX.
 17. A method of reducing the toxicity of, orreducing side effects associated with, docetaxel (DTX), or formulationof DTX, or of reducing hypersensitivity in a subject upon treatment withDTX or a formulation of DTX, comprising administering a macromoleculeaccording to claim
 1. 18. The method of claim 17, wherein: (a) thetoxicity that is reduced is selected from the group consisting ofhematologic toxicity, neurological toxicity, gastrointestinal toxicity,cardiotoxicity, hepatotoxicity, nephrotoxicity, and encephalotoxicity,or (b) the side effects which are reduced are selected from the groupconsisting of neutropenia, leukopenia, thrombocytopenia, myelotoxicity,myelosuppression, neuropathy, fatigue, vertigo, encephalopathy, anemia,dysgeusia, dyspnea, constipation, anorexia, nail disorders, fluidretention, asthenia, pain, nausea, vomiting mucositis, alopecia, skinreactions, myalgia, hypersensitivity and anaphylaxis.
 19. A compound, ora salt thereof, comprising a 2,2′-thiodiacetic acid linker covalentlyattached to the hydroxy group of the propanoyl side chain of DTX. 20.The compound of claim 19, or a salt thereof, having the structure:


21. The compound of claim 19, further comprising a lysine residuecovalently attached by its first amine to the distal carboxy group ofthe 2,2′-thiodiacetic acid linker; and by its second amine to a PEG.